**1. Introduction**

The study of the contribution of human genetics to the risk of severe malaria has a long history, with Haldane in the 1950s reporting a major role of the sickle cell mutation (HbS), in the protection against severe disease (Haldane, 1949). Since then, genetic variants of βglobin (HbE: Hutagalung et al., 1999; HbC: Agarwal et al., 2000; HbS: Aidoo et al., 2002), α-globin (Mockenhaupt et al., 2004), Band 3 protein (Foo et al., 1992), HLA (Hill et al., 1991) and several cytokine loci (Tumor Necrosis Factor-alpha: Knight et al., 1999; Interleukin-12: Morahan et al., 2002a; Interferon-alpha receptor-1: Aucan et al., 2003; Interleukin-4: Gyan et al., 2004) have been demonstrated to confer protection to severe malaria. To date, the majority of studies have been case/control association studies, comparing severe malaria to uncomplicated cases. Due to the fact that *Plasmodium falciparum* is the etiological agent of severe malaria, all studies have consequently focussed on this parasite. The other congeneric species and notably *Plasmodium vivax*, can, however, cause severe disease, albeit at a much lower incidence, and warrant an increased research effort (Price et al., 2007).

Here we argue that for infectious diseases in general and for malaria parasites in particular, more attention should be paid to the "biological" course and outcome of infection in addition to severe disease. This is for several reasons: (i) the majority of infections in endemic settings do not cause severe disease. Indeed, severe disease is a collective term that englobes multiple pathologies (including cerebral malaria, severe malaria anaemia, metabolic acidosis, multiple organ failure) that likely involve very different biological pathways and thus should not be analysed as a single phenotype; (ii) the progression from clinical malaria to asymptomatic infection defines the acquisition of clinical immunity and identifying the mechanisms underlying this tipping point is central to the development of disease control methods; (iii) *P. falciparum* is remarkable in that sterilising anti-parasite immunity is never achieved. Although the parasite has a variety of mechanisms enabling this, the human also plays a part. Identifying pathways that "enable" the parasite to persist without elimination will provide insight into this apparent immune defence dysfunction; (iv) the biology of the parasite within the host will be informative as to how the parasite

Human Genetic Contribution to the Outcome of Infection with Malaria Parasites 269

The malaria parasite spp. lifecycles will undoubtedly be known to readers or covered in associated chapters. Here, we place the life-cycle within a perspective useful for human genetic studies. Although differing in the details, different *Plasmodium* spp. broadly share three distinct life cycle parts within the human host: invasion and asexual proliferation within the liver, invasion and asexual proliferation within red blood cells and the production of sexual stages, gametocytes, from a proportion of these asexually proliferating haploid parasites within the red blood cells. These gametocytes are essential for successful transmission to mosquitoes and subsequent infection of new human hosts. The parasite, as with any other sexually reproducing eukaryote, will, to the best of its capacity, have evolved to optimally exploit its host and maximise its reproductive rate through infection of new hosts. In turn, the human is expected to have evolved to minimise the damage caused by the parasite. The course of infection and the outcome of the human-parasite interaction are thus quantifiable by measurement of the density of asexual and sexual circulating parasites and

Placing the in-host biology of the parasite within the context of the clinical outcome of the infection is central to unravelling how human genetics impacts upon the pathophysiology of malaria. The clinical outcome of infection ranges from severe through mild disease to asymptomatic infection. Less well documented is the progression of clinical expression during the course of a single infection. Early treatment, thanks to considerable public health efforts, has now reduced the burden of disease and in study cohorts we do not know whether an individual with mild malaria would have progressed to severe disease if left untreated and/or eventually control but not eliminate the infection, leading to a chronic long-term asymptomatic infection. Thus, our focus is on parasite biological phenotypes in the context of symptomatic or asymptomatic infection outcomes, with no division into mild *vs*. severe disease. Thus, we ask why is there variation in the density of asexual parasite stages that individuals can withstand before becoming symptomatic and once symptomatic, why do only some infections attain very high densities. Transmission is a crucial part of the lifecycle for the parasite and there is good evidence that the parasite has evolved to optimise its transmission to mosquitoes with respect to the host response to infection (Paul et al., 2003). We thus examine the human factors that influence gametocyte production and whether they differ in symptomatic and asymptomatic infections. Some biological phenotypes, most notably those pertaining to the exo-erythrocytic stages, are beyond our current ability to measure in sufficient detail but do warrant increased research effort. Preventing liver stage infection and eliminating latent hypnozoites in relapsing species are

Major differences in certain life-cycle aspects do exist amongst the *Plasmodium* spp. infecting man and surprisingly little is known about the biology or the acquired immune response to species other than *P. falciparum*. The major apparent differences include the capacity to form relapsing latent hypnozoite stages that reside in the liver (*P*. *vivax* and *Plasmodium ovale*), the rate of development of the asexually replicating erythrocytic stages (48hours for *P. falciparum*, *P. vivax* and *P. ovale vs*. 72 hours for *P. malariae*), the capacity for asexual stage parasites to cytoadhere (*P. falciparum*) and the predilection for invading red blood cells of

**2. Malaria parasite course and outcome of infection** 

**2.1 Quantitative malaria-related phenotypes** 

the frequency of clinical episodes.

clear targets for reducing the prevalence of infection.

optimises its exploitation of and subsequent transmission from the human host. The parasite must persist and transmit to mosquitoes in the face of very differing immune environments. The differential impact of human genetics according to the clinical outcome of infection will throw light on how the parasite manages its strategy for survival and reproduction (transmission).

Focussing on infection enables implementation of a family-based study design that controls for population sub-structure and admixture. Such an approach would be impractical for the study of severe disease because of its relative infrequency. Longitudinal family-based studies enable a more detailed real-time analysis of the human response to infection. Thus, as well as controlling for population sub-structure, they can (i) reveal how the same individual responds at different times in his life and thus generate insight into the acquisition of clinical and anti-parasite immunity; (ii) enable incorporation of parasite genetics both with respect to the long-term co-evolutionary trajectory of the host-parasite duo and the short-term impact of intervention (Loucoubar et al., 2011a).

Evidence for a contribution of host genetic factors to mild clinical malaria and biological phenotypes, such as number of clinical episodes, parasite density, immune responses to *P. falciparum* antigens has progressed with the development of increasingly sophisticated techniques. Population level differences in susceptibility to malaria have been observed between sympatric ethnic groups (Modiano et al., 1996) and, at a finer scale, differential phenotypic expression was observed in monozygotic and dizygotic twins; there was greater phenotypic similarity in monozygotic twins, strongly suggesting genetic control as such twins are genetically more similar than dizygotic twins (Jepson et al., 1995). Segregation studies that assess the extent of phenotypic similarity in families demonstrated cosegregation of parasite density and of prevalence of mild malaria in families (Rihet et al., 1998a). Microsatellite typing of family-based cohorts enabled this segregation to be narrowed down to chromosomal regions (Flori et al., 2003; Garcia et al., 1998; Rihet et al., 1998b; Sakuntabhai et al., 2008; Timmann et al., 2007). Candidate gene approaches have also shown association of specific genetic polymorphisms with mild clinical malaria (Kun et al., 2001; Walley et al., 2004; Williams et al., 1996). Emphasis has understandably been placed on clinical malaria and very few studies have considered asymptomatic malaria and only to a limited extent (Flori et al., 2003; Garcia et al., 1998; Mombo et al., 2003; Rihet et al., 1998a, 1998b; Timmann et al., 2007).

This chapter presents a summary of the achievements in the field of genetic analysis to date, the benefits of examining biological parasite phenotypes and the practical aspects of sampling and analysis. Firstly, we discuss in some detail issues concerning phenotype choice, the pros and cons of case/control vs. family based methods, the importance of context-dependency and of taking into account covariates. We then expand upon the utility of heritability analyses and describe the novel methods that should be a requisite for performing a genetic analysis of quantitative malaria parasite phenotypes. We then discuss our own findings using heritability and genome wide analyses that have led us to propose a novel hypothesis concerning the role of allergy in malaria. Finally we outline the future direction that genetic studies should take, most notably concerning the need to develop tools to examine gene-gene and gene-environment interactions.
