**6. Linkage analyses of malaria phenotypes identifies regions associated with atopy**

Linkage analyses are the classical statistical genetic analyses testing for co-segregation between a chromosomal region and a phenotype of interest. Prior to the high density genotyping now available for genome wide association, linkage analysis was the preferred method for gene discovery, enabling chromosomal regions of interest to be identified with relatively low genotyping coverage.

#### **6.1 Genome wide linkage analysis of** *P. falciparum* **in Senegal**

Our genome wide linkage analysis in the two Senegalese cohorts identified three novel regions of linkage in addition to the 5q31 region that has been previously reported to be linked to asymptomatic parasite density (Flori et al., 2003; Garcia et al., 1998; Rihet et al., 1998b). All of the regions have been previously found to be linked to asthma/atopic disease or related phenotypes, such as IgE titres (Iyengar et al., 2001; Jang et al., 2005; Kurz et al., 2006; Xu et al., 2001; Zhang et al., 2003). The novel regions of linkage were chromosome 5p15-p13 and 13q13-q22 linked with the number of *P. falciparum* clinical malaria attacks in Dielmo, and chromosome 12q21-q23 with the maximum parasite density during asymptomatic carriage in Ndiop. The linkage results differ for the two cohorts, likely reflecting the important differences both in the ethnic backgrounds and in the prevailing transmission conditions. Interestingly, the 5q31 locus has been linked to several immune related disorders, including asthma/atopy (Meyers et al., 1994), inflammatory bowel disease (Lee et al., 2002), Crohn disease (Peltekova et al., 2004), Celiac disease (Latiano et al., 2007) and psoriasis (Friberg et al., 2006). Moreover, genes within the 5q31 locus have been suggested to regulate delayed-type hypersensitivity responses associated with Leishmania chagasi infection (Jeronimo et al., 2007). This region contains a cluster of cytokines, among which IL12B may play a critical role since it has been associated with some immune-related diseases. An insertion/deletion polymorphism in the promoter region of IL12B has been reported to be associated with psoriasis (Cargill et al., 2007) and cerebral malaria (Morahan et al., 2002a) while two intronic SNPs were associated with asthma (Morahan et al., 2002b; Randolph et al., 2004).

With the exception of the β-globin locus, there was no overlap of the regions of linkage that we detected and the location of the genes that have been previously reported to be associated with severe/cerebral malaria. This apparent discordance between genes

Human Genetic Contribution to the Outcome of Infection with Malaria Parasites 283

et al., 2000). Finally, *P. falciparum* produces translationally controlled tumor protein, which is a homolog of the mammalian histamine-releasing factor that causes histamine release from human basophils (MacDonald et al., 2001). How this could benefit the parasite is not known, but the vasodilatory effects of histamine might permit the parasites to circulate more readily and histamine might increase endothelial cell-surface expression of thrombomodulin, which is both a tissue anticoagulant and a receptor for parasitized erythrocyte sequestration.

Our heritability, correlation and genome wide linkage study results are consistent with there being a relationship between malaria and allergy and raise the hypothesis that the development of clinical malaria may be due to an allergic reaction to malaria parasites or byproducts of parasite infection, or that allergy/atopy and the response to malaria infection may share common mechanisms. Thus, clinical immunity to malaria may indeed be immunotolerance and absence of allergic-type responses rather than the presence of neutralising antibodies to malaria "toxins" as previously suggested (Jakobsen et al., 1995). Several lines of additional evidence support the concept that susceptibility to malaria and atopy may be related to the same immunological defect. In Ethiopia, atopic children had a higher prevalence of malaria attacks (Haileamlak et al., 2005), while in Tanzania maternal malaria had a protective effect on wheezing in children age of four (Sunyer et al., 2001). Finally, a mouse model for human atopic disease (NC/Jic) was found to be susceptible to murine malaria (Ohno et al., 2001) and a major quantitative trait locus (derm1) for atopic

disease mapped close to the region controlling parasitemia (Kohara et al., 2001).

Most of the protective variants are thought to have emerged in populations living in regions endemic for malaria as a result of the high selective pressure due to the parasite (Kwiatkowski, 2005). The past decade has seen growing evidence of ethnic differences in susceptibility to malaria and of the diverse genetic adaptations to malaria that have arisen in different populations. The fact that different malaria-resistance alleles have arisen in different places suggests that a great deal of selection by malaria has happened relatively recently in human history and certainly since human migration out of Africa (Eid et al., 2010). Such population differences in susceptibility to malaria are becoming more amenable to study since the development of high through-put genetic technology, thereby allowing us

Association studies are used for identifying genes and their common allelic variants involved in predisposition to a disease. Such studies are performed after localization of susceptible loci by linkage analysis. This method compares the allele frequency of a genetic marker of affected and non-affected individuals, chosen at random in a population (casecontrol study). The marker might be the causal polymorphism or any polymorphism in linkage disequilibrium (LD) with the causal one. A positive association with one marker suggests that this marker is in LD with the causal polymorphism. The LD between two markers is defined by the existence of a combination of alleles of these markers more often than expected by chance. The choice of the control population is one of the most important problems of case-control studies: if the control group is not from the same population as the affected individuals, uncontrolled environmental factors or population stratification might

**7. Genetic association studies and the post-genomic era** 

to genetically dissect the outcome of infection.

**7.1 Candidate and genome wide association studies** 

responsible for severe malaria and those controlling the response to *Plasmodium* infection in our study may also indicate that the mechanisms (and genes) involved in the protection against severe malaria are largely independent of those involved in the response to mild clinical malaria and/or the control of blood parasitemia.

#### **6.2 Role of allergy in malaria**

The acquisition of immunity to the human lethal malaria parasite *P. falciparum* develops very slowly and is not sterilising. Even in zones where the transmission intensity is high, the development of immunity only results in a premunition leading to a reduction in the number of clinical episodes and the progressive control of parasite density. Cytophilic immunoglobulins (IgG1 & IgG3), which are capable of eliminating the parasites by opsonisation, play an important role in this premunition (Wilson & McGregor, 1973). An important role of the Th1/Th2 balance in the development of clinical malaria has been suggested by numerous studies (e.g. Elghazali et al., 1997). Orientation of the immune response towards a Th1 versus a Th2 profile, will respectively promote IgG *vs*. IgE proliferation. The role of IgE in clinical and severe malaria is still poorly documented and results are controversial. *P. falciparum*-specific IgE is elevated in malaria patients and has been proposed to play a pathogenic role in severe malaria (Elghazali et al., 1997; Perlmann et al., 1997), although high levels in individuals with asymptomatic infections were associated with protection (Bereczky et al., 2004). The induction of a Th2 biased immune response by *P. falciparum* may generate a tendency to develop a Th2 type immune response to other antigens. Dendritic cells that are oriented to a Th2 phenotype by an antigen are more susceptible to orient the immune response towards a Th2 profile when confronted by a second antigen (de Jong et al., 2002). It has been suggested that the Th2 bias induced by *P. falciparum* may exacerbate allergy and explain the higher than normal frequency of several cancers in malaria endemic populations (Taylor-Robinson, 1998).

Direct evidence has been found for a pathogenic role of histamine (a major effector molecule in allergy response) in mouse malaria models using both genetic and pharmacological approaches; histamine binding to Histamine receptor-1 (H1R) and receptor-2 (H2R) increases the susceptibility of mice to infection with *Plasmodium* and histidine decarboxylase–deficient mice, which are free of histamine were highly resistant to severe malaria (Beghdadi et al., 2008). H1R mediates most of the proinflammatory effects of histamine (Bryce et al., 2006). The anti-inflammatory and immunosuppressive effects of histamine are largely dependent on stimulation of H2R. In addition, there is suggestion that histamine might influence the polarization of T-helper cell development through inhibitory effects on dendritic cells (Idzko et al., 2002). Reports indicate that specific components of the innate immune system, including eosinophils (Kurtzhals et al., 1998), basophils (Nyakeriga et al., 2003), and Mast cells (MCs) (Furuta et al., 2006), could play important roles in the pathogenesis of malaria. Increased levels of histamine in plasma and tissue, derived from basophils and MCs, notably following stimulation by IgE through the high affinity receptor FcεR1, are associated with the severity of disease in humans infected with *P. falciparum* and in animal malaria models (Bhattacharya et al., 1988; Srichaikul et al., 1976). Chlorpheniramine, a histamine receptor-1 agonist reversed resistance to chloroquine and amodiquine both in vivo and in vitro (Sowunmi et al., 2007). Moreover, astemizole, another HR1 agonist, was identified as an antimalarial agent in a clinical drug library screen (Chong

responsible for severe malaria and those controlling the response to *Plasmodium* infection in our study may also indicate that the mechanisms (and genes) involved in the protection against severe malaria are largely independent of those involved in the response to mild

The acquisition of immunity to the human lethal malaria parasite *P. falciparum* develops very slowly and is not sterilising. Even in zones where the transmission intensity is high, the development of immunity only results in a premunition leading to a reduction in the number of clinical episodes and the progressive control of parasite density. Cytophilic immunoglobulins (IgG1 & IgG3), which are capable of eliminating the parasites by opsonisation, play an important role in this premunition (Wilson & McGregor, 1973). An important role of the Th1/Th2 balance in the development of clinical malaria has been suggested by numerous studies (e.g. Elghazali et al., 1997). Orientation of the immune response towards a Th1 versus a Th2 profile, will respectively promote IgG *vs*. IgE proliferation. The role of IgE in clinical and severe malaria is still poorly documented and results are controversial. *P. falciparum*-specific IgE is elevated in malaria patients and has been proposed to play a pathogenic role in severe malaria (Elghazali et al., 1997; Perlmann et al., 1997), although high levels in individuals with asymptomatic infections were associated with protection (Bereczky et al., 2004). The induction of a Th2 biased immune response by *P. falciparum* may generate a tendency to develop a Th2 type immune response to other antigens. Dendritic cells that are oriented to a Th2 phenotype by an antigen are more susceptible to orient the immune response towards a Th2 profile when confronted by a second antigen (de Jong et al., 2002). It has been suggested that the Th2 bias induced by *P. falciparum* may exacerbate allergy and explain the higher than normal frequency of several

Direct evidence has been found for a pathogenic role of histamine (a major effector molecule in allergy response) in mouse malaria models using both genetic and pharmacological approaches; histamine binding to Histamine receptor-1 (H1R) and receptor-2 (H2R) increases the susceptibility of mice to infection with *Plasmodium* and histidine decarboxylase–deficient mice, which are free of histamine were highly resistant to severe malaria (Beghdadi et al., 2008). H1R mediates most of the proinflammatory effects of histamine (Bryce et al., 2006). The anti-inflammatory and immunosuppressive effects of histamine are largely dependent on stimulation of H2R. In addition, there is suggestion that histamine might influence the polarization of T-helper cell development through inhibitory effects on dendritic cells (Idzko et al., 2002). Reports indicate that specific components of the innate immune system, including eosinophils (Kurtzhals et al., 1998), basophils (Nyakeriga et al., 2003), and Mast cells (MCs) (Furuta et al., 2006), could play important roles in the pathogenesis of malaria. Increased levels of histamine in plasma and tissue, derived from basophils and MCs, notably following stimulation by IgE through the high affinity receptor FcεR1, are associated with the severity of disease in humans infected with *P. falciparum* and in animal malaria models (Bhattacharya et al., 1988; Srichaikul et al., 1976). Chlorpheniramine, a histamine receptor-1 agonist reversed resistance to chloroquine and amodiquine both in vivo and in vitro (Sowunmi et al., 2007). Moreover, astemizole, another HR1 agonist, was identified as an antimalarial agent in a clinical drug library screen (Chong

clinical malaria and/or the control of blood parasitemia.

cancers in malaria endemic populations (Taylor-Robinson, 1998).

**6.2 Role of allergy in malaria** 

et al., 2000). Finally, *P. falciparum* produces translationally controlled tumor protein, which is a homolog of the mammalian histamine-releasing factor that causes histamine release from human basophils (MacDonald et al., 2001). How this could benefit the parasite is not known, but the vasodilatory effects of histamine might permit the parasites to circulate more readily and histamine might increase endothelial cell-surface expression of thrombomodulin, which is both a tissue anticoagulant and a receptor for parasitized erythrocyte sequestration.

Our heritability, correlation and genome wide linkage study results are consistent with there being a relationship between malaria and allergy and raise the hypothesis that the development of clinical malaria may be due to an allergic reaction to malaria parasites or byproducts of parasite infection, or that allergy/atopy and the response to malaria infection may share common mechanisms. Thus, clinical immunity to malaria may indeed be immunotolerance and absence of allergic-type responses rather than the presence of neutralising antibodies to malaria "toxins" as previously suggested (Jakobsen et al., 1995). Several lines of additional evidence support the concept that susceptibility to malaria and atopy may be related to the same immunological defect. In Ethiopia, atopic children had a higher prevalence of malaria attacks (Haileamlak et al., 2005), while in Tanzania maternal malaria had a protective effect on wheezing in children age of four (Sunyer et al., 2001). Finally, a mouse model for human atopic disease (NC/Jic) was found to be susceptible to murine malaria (Ohno et al., 2001) and a major quantitative trait locus (derm1) for atopic disease mapped close to the region controlling parasitemia (Kohara et al., 2001).
