Polymorphism of immune genes

Genetic diversity in a number of different immune genes involved in our response to malaria infection have been associated with disease susceptibility, the range of genes involved reflecting the complexities of our immunological battle with the malaria parasite (see Fig. 13.3). Notable among these studies was the early association found with particular MHC class I molecules, glycopro-teins that recognize the parasite within infected cells, delivering the foreign protein to the cell surface to be presented to CD8+ cytotoxic T lymphocytes (Box 12.1). The malaria parasite replicates in human liver cells before invading red blood cells. It is in these liver cells that MHC class I molecules can act as they are not expressed in red blood cells.

Possession of a specific human leukocyte antigen HLA-B53 was found to be associated with protection from severe malaria in a large case-control study of children in The Gambia (Hill et al. 1991). In this West African population about 1% of children less than 5 years of age die from malaria, and 20% of people carry P. falciparum in their blood. Hill and colleagues studied 619 children with severe malaria, initially analysing 45 different class I antigens among half the cases and finding association with the HLA-B53 on comparison with different control groups including children with mild non-malarial illness, mild malaria or severe non-malarial illness, and healthy adult blood donors. Given that the association may have arisen by chance the investigators then analysed the remaining cases only for HLA-B53, this time genotyping the allele directly by a PCR based method. Again significant association was found with a frequency of 16.9% among cases of severe malaria and 25.4% among mild controls with non-malarial illness, and similar frequencies in the other control groups. Taken together the data demonstrated protection from severe malaria associated with possession of HLA-B53 with an odds ratio of 0.59 (0.43-0.81, P = 0.008) (Hill et al. 1991).

The degree of protection associated with having HLA-B53 was not as marked as for possession of the sickle cell trait but, as the latter is less common in this population, they both appear to contribute a similar level of protection. Each variant has been estimated to prevent about one in ten potential cases of severe malaria. HLA-B53 is particularly common in West Africa with a frequency of 40% in Nigeria and 25% in The Gambia; allele frequencies are lower in other parts of Africa (for example 2% in South Africa) while in Caucasians and South East Asian populations the allele is rare (0-1%) and the allele is absent in Pacific regions (Hill et al. 1991). Association was also found with protection from severe malaria for an HLA-DRB1 variant encoding a specific class II allele, DRB1*1302-DQB1*0501, with the effect specific to protection from severe malarial anaemia (Hill et al. 1991). As noted in Chapter 12 and elsewhere, the extensive linkage disequilibrium present in the MHC makes fine mapping such disease associations and assigning causality to specific variants a major challenge.

Insights into the molecular events occurring when HLA-B53 binds to P. falciparum were gained through understanding the structure of the molecule using X-ray crystallography studies. These revealed the architecture of a specific pocket on the molecule critical to recognition of the parasite found on many HLA-B alle-les. Within this groove, bound water molecules, acting in concert with the side chains of polymorphic residues, provide functional malleability, which enables high affinity/low specificity binding of multiple peptide epitopes (Smith et al. 1996). In the specific case of HLA-B53, the peptide binding groove was observed to be widened (Fig. 13.9). This may be part of the answer to why this particular allele should be associated with protection from severe malaria.

The malaria parasite is not an idle bystander in this process of interaction with HLA-B53. HLA-B53 binds to a specific fragment of P. falciparum, the circumsporozoite protein, leading to an attack by cytotoxic T lymphocytes (CTLs) on the parasite. Four variants of the parasite are found in The Gambia that differ in their circumsporozoite protein, of which two, the cp26 and cp29 variants, bind HLA-B53. Together these variants appear to jam the mechanism by which an interaction between HLA-B53 and parasite circumsporozoite protein would normally lead to CTL attack. In experiments, the presence of one variant renders the CTLs unable to kill the parasites carrying the other variant. Moreover, these two variants occur together in infected patients' blood much more often than expected. Remarkably, it appears that each individually suppresses the CTL response to the other. How this occurs is unclear but it seems cooperation between the two parasite strains leads to a mutual survival advantage. This is thought to represent a successful immune evasion strategy by the parasite; selective pressures can clearly act in both directions (Gilbert et al. 1998).

Genes encoding other key players in the body's immune response to malarial infection also show evidence of genetic variation associated with disease susceptibility, including innate and adaptive immunity (see Fig. 13.3) (Kwiatkowski 2005). For example, the CD40 ligand is an important component of the immune response, involved in B cell proliferation, activation of antigen presenting cells, and regulation of immunoglobulin class switching. Rare variants of CD40LG (also known as TNFSF5) encoding the CD40 ligand at chromosome Xq26 are associated with X-linked hyper-IgM syndrome (OMIM 308230), a rare immunodeficiency disorder associated with severe bacterial and life threatening infection. A single nucleo-tide polymorphism (SNP) in the CD40LG promoter has been associated with resistance to severe malaria,

Figure 13.9 Binding by an HLA-B53 molecule to a peptide from Plasmodium falciparum. (A) Parasite peptide (indicated by an arrow) in the HLA binding groove of the molecule. (B) Interaction viewed from above. Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Genetics (Cooke and Hill 2001), copyright 2001.

Figure 13.9 Binding by an HLA-B53 molecule to a peptide from Plasmodium falciparum. (A) Parasite peptide (indicated by an arrow) in the HLA binding groove of the molecule. (B) Interaction viewed from above. Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Genetics (Cooke and Hill 2001), copyright 2001.

showing evidence of selection on haplotype analysis (Sabeti et al. 2002a, 2002b). Differences in antibody levels against malarial antigens have been found among particular ethnic groups such as the Fulani in West Africa, who show increased resistance to malaria compared to other ethnic groups (Box 13.3).

Genes encoding many different mediators of the inflammatory response have been studied in relation to malaria including pro- and anti-inflammatory cytokines such as tumour necrosis factor (TNF), IL-1, and IL-10, as well as mediators such as IFNy and the gene encoding inducible nitric oxide (NO) synthase, responsible for the important free radical NO implicated in the pathogenesis of cerebral malaria (see Fig. 13.3) (Rockett et al. 1991; Clark et al. 2003b; Kwiatkowski 2005). Many of these and other studies of genetic factors determining malaria susceptibility have been based on a candidate gene approach for genetic association and in a number of cases conflicting results have been obtained on replication studies (Section 2.4).

In part this reflects the many potential caveats in such analyses inherent to the study of common disease but also some specific to malaria and the genetically diverse populations often studied in this disease. African populations for example show high levels of genetic variation with specific and distinct patterns of linkage disequilibrium between populations, which offer greater potential for resolution of specific functional alleles but also more capacity for apparent fail to replicate using a given set of genetic markers. Differences in malarial transmission intensities and prevailing parasite diversity will also vary between locations, which can be highly significant when considering specific disease phenotypes (Kwiatkowski 2005).

As an example to consider in more detail, genetic diversity at or near to TNF, the gene encoding TNF, has been associated with susceptibility to severe malaria by a number of studies. TNF is one of the earliest genes to be expressed in response to infection and acts to initiate and orchestrate the inflammatory cascade fighting infection, however dysregulation of TNF production can itself cause disease. In septic shock, for example, there is good evidence that TNF has a causative role as it is produced during the septic shock syndrome, it causes the development of the syndrome when given to uninfected animals, and neutralizing TNF in infected septic animals prevents

Box 13.3 Resistance to malaria among the Fulani

The Fulani people living in rural savannah areas of Burkina Faso and Mali in West Africa were found to be more resistant to malarial infection with Plasmodium falciparum than other sympatric ethnic groups living in the same hyperendemic areas for malaria transmission such as the Mossi and Rimaibe (Modiano et al. 1996; Dolo et al. 2005). Despite similar exposure rates, markedly lower levels of parasitaemia, fewer episodes of fever or clinical infection, and less severe disease were found among the Fulani. The Fulani are genetically distinct from the other two groups (Modiano et al.

2001a) but no known malaria risk alleles such as Hb S, Hb C, thalassaemia, G6PD, or HLA-B53 could be found to account for the observed differences in disease resistance (Modiano et al. 2001b). The antibody response to malarial antigens was found to be increased among the Fulani with evidence that genetic variation in the IL4 gene encoding inter-leukin-4 was associated with increased antibody levels (Luoni et al. 2001). Gene expression profiling suggests that a functional deficit of T regulatory cells may lead to the observed higher resistance to malaria among the Fulani (Torcia et al. 2008).

the development of shock (Tracey and Cerami 1993). Similarly in cerebral malaria, there is growing evidence that while TNF protects against infection, in excess it can directly cause disease (Kwiatkowski et al. 1990). Genetic polymorphism at the TNF locus has been associated with susceptibility to many infectious, autoimmune, and inflammatory diseases although many studies appear conflicting and specific functional variants remain hard to localize due to coinheritance of genetic variants across the locus (Section 2.4.4).

There is evidence to implicate TNF with disease outcome from malarial infection based on linkage studies in The Gambia and Burkina Faso (Jepson et al. 1997; Flori et al. 2003) and using a candidate gene approach. Children with severe malaria had a higher allele frequency of a particular SNP in the promoter region of the TNF gene, suggesting a role in disease susceptibility (McGuire et al. 1994). Individuals homozygous for the A' allele of rs1800629 (located 308 bp 5' of the transcriptional start site and known as 'TNF-308') showed a seven-fold increased risk of cerebral malaria in a large case-control study in The Gambia. Association was also reported with more rapid symptomatic reinfection with P. falciparum in Gabon (Meyer et al. 2002), with severe malaria and other infectious diseases in Sri Lanka (Wattavidanage et al. 1999), and with malaria morbidity in Kenya (Aidoo et al. 2001) and some evidence of association with parasitaemia in Burkina Faso (Flori et al. 2005). Whether this specific SNP or linked alleles are functionally important remains controversial (Section 2.4.4).

Other TNF promoter SNPs have shown evidence of disease association, for example with severe malarial anaemia (rs361525) (McGuire et al. 1999), while a rare variant more distally in the promoter (rs1800750, 'TNF-376') region showed association with severe malaria in The Gambia and Kenya (Knight et al. 1999). The latter was identified as a candidate functional variant based on analysis of sites of protein-DNA interaction using the DNA footprinting technique. A single nucleotide substitution was found to modulate the binding of a specific transcription factor, Oct-1, which showed higher affinity to the rs1800750 'A' allele and was associated with increased gene expression in human monocytes (Knight et al. 1999). As elsewhere in the MHC there is extensive linkage disequilibrium across the TNF locus and association with malaria and other infectious disease have been found at the neighbouring gene LTA encoding lymphotoxin alpha. Here a putative functional variant was resolved by analysis of haplotype-specific differences in gene expression with a specific SNP (rs2239704, 'LTA+80'), shown by haplotype-specific chromatin immunoprecipitation (ChIP) (Section 11.5.3) to modulate recruitment of the transcriptional repressor activated B cell factor 1 (Knight et al. 2003, 2004). The low producer allele defined by rs2239704 was subsequently associated with reduced parasitaemia in a study in Burkina Faso (Barbier et al. 2008) and with early onset leprosy in a study analysing individuals from Vietnam, Brazil, and India (Alcais et al. 2007). Whether this SNP explains some of the reported TNF associations or indeed is a marker for a further unidentified functional variant remains to be fully resolved, but what is clear is that there is a strong signal of association at the TNF locus with malaria and other infectious diseases.

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