The pathophysiological basis of SMA is complex and poorly understood. One method for exploring the genes and gene pathways that condition susceptibility to SMA is to utilize a candidate gene approach for both cross-sectional and longitudinal investigations. This approach allows for an increased understanding of the mechanisms that confer protection against development of SMA once children become infected with falciparum malaria (acute disease), and also provides information about how polymorphic variants condition susceptibility to SMA and/or mortality throughout the critical phases of naturally-acquired immunity (longitudinal outcomes). We hypothesized that variation at rs3212227 locus may provide important information about the role of IL-12 in protecting against SMA.
Children with falciparum malaria underwent extensive clinical evaluations to rule out additional causes of anaemia so that they could be appropriately controlled for in the multivariate models. For example, since HIV-1 and bacteremia are some of the most common anaemia-promoting infectious diseases in African children [27–29], all study participants were tested for these co-pathogens. It is important to note that children exposed to HIV-1, yet lack definitively identifiable virus in their system, also have enhanced severity of anaemia when they acquire falciparum malaria . Unlike previous studies, the multivariate models therefore controlled for HIV-1 exposure, HIV-1 positivity, and bacteremia. Since severe anaemia is the primary manifestation of severe malaria in holoendemic P. falciparum transmission regions, with cerebral malaria being a rare occurrence, this study specifically examined the role of rs3212227 in conditioning the development of severe anaemia and as such, it remains to be determined if results obtained here are applicable to areas with differing endemicities of malaria in which individuals develop severe disease manifestations apart from severe anaemia.
IL-12 plays a critical role in modulating the immune response by promoting CD4+ T cells and NK cells to differentiate towards a Th1 phenotype for production of IFN-γ, an important component of cell-mediated immunity that helps eliminate intracellular pathogens . Accumulating evidence shows that IL-12 is a critical factor for augmenting protective immune responses against malaria in animal models [7, 32, 33]. Our previous studies showing that reduced IL-12p70 production is associated with enhanced severity of malaria in African children [8, 34] illustrate the importance of IL-12 in human malaria as well. Consistent with these investigations, the current study demonstrated that children with acute malaria that develop SMA have significantly lower circulating IL-12p40/p70 levels than children that fail to progress to severe anaemia (i.e., the non-SMA group).
Previous examination of the rs3212227 polymorphism in Tanzanian children demonstrated that homozygous C individuals had higher mortality from cerebral malaria, whereas no significant associations were found between rs3212227 genotypes and severe malaria (defined as a mixed phenotypic outcome) in children from coastal Kenya . Our recent study in Thai adults with falciparum malaria showed that although carriage of the rs3212227 CC genotype was enriched in the severe disease group, multivariate modelling did not reveal a significant association between the genotype and disease severity . In addition, a comprehensive longitudinal study in an adjacent region to where the current study was conducted utilized fifty-five tagging single nucleotide polymorphisms (covering genes encoding IL12A, IL12B, IL12RB1, and IL12RB2) to demonstrate a number of important associations between genetic variation and SMA, of which rs3212227 did not emerge as significant . However, results presented here clearly demonstrate that homozygosity at the C allele was associated with an increased risk of developing SMA in children with acute disease, while having no effect on longitudinal outcomes (i.e., SMA and mortality) over three years of follow-up. While the reason for differences in the predominately Luo ethnic populations examined in both studies is unclear, it may be attributable to the fact that we controlled for the influence of common paediatric infectious diseases, such as HIV-1 and bacteremia, that influence both anaemia and mortality throughout childhood. Furthermore, the distribution of genotypes in the combined clinical groups (non-SMA and SMA) displayed significant departure from HWE, possibly resulting from selection at the rs3212227 SNP due to 'enrichment' of malaria-infected individuals in the current study. Although not available for the current study, examination of the genotypes in a large number of non-malaria infected 'controls' would help to determine if the HWE deviation at rs3212227 observed in the current study is indeed due to malaria-mediated selection.
Since 3'UTRs contain regulatory elements that are highly conserved and control translational activity, variation within the 3'UTR can affect transcriptional stability and binding of translational proteins . A previous investigation examining the rs3212227 in Turkish adults showed that individuals with the CC genotype produced the highest levels of IL-12p70 in cultured peripheral blood mononuclear cells (PBMC) [22, 36], while another study in two ethnic groups from Bulgaria revealed that the AA genotype was associated with the highest PBMC IL-12p40 production [22, 36]. However, recent studies in individuals with lepromatous leprosy and Chagas' disease failed to find any significant relationships between rs3212227 genotypes and IL-12p70 production [20, 21]. Consistent with these investigations, stratification of children in the current study, according to genotypes, failed to show any significant differences in circulating IL-12p40/p70 levels across the groups. Moreover, examination of IL-12p40/70 in cultured PBMC from children with malaria in the current study also failed to show any significant association with rs3212227 genotypes (data not presented). The lack of a functional association between rs3212227 variants and IL-12 production reported here is supported by our in silico analysis (using the web site http://www.cbil.upenn.edu/cgi-bin/tess/tess) which revealed that the region encompassing the rs3212227 had no major transcription factor binding site changes that would be affected by SNP variation within this region. Although the exact reason for the lack of a functional association between rs3212227 genotypes and IL-12p40/p70 levels reported here remains to be determined, it is possible that additional polymorphic variants, such as IL12Bpro , or variation in other genes that influence IL-12 production (e.g. IL-10 and IL-4) may be responsible for mediating IL-12 generation [36, 37].
Since the mechanism through which IL-12 provides protection against malaria in murine models appears to be related to the ability of IL-12 to promote IFN-γ and tumour necrosis factor (TNF)-α production that, in turn, provide enhanced NO generation , we examined the association between the rs3212227 variants and all three of these inflammatory mediators. However, circulating levels of IFN-γ, TNF-α, or NO were not significantly different upon stratifying into genotypic groups (data not presented).
Recent data from our laboratory, obtained in the same population of children, demonstrated that multi-site loci provide more informative information about malarial disease outcomes than information obtained for a single locus [39, 40]. These studies further revealed that combinations of functional polymorphic alleles in haplotypes interact to modulate the individual effects of SNPs and variable number tandem repeats (VNTRs) [39, 41]. As such, we are currently expanding the current study to include additional polymorphisms in the IL12B gene (and other immunoregulatory genes that influence IL-12), to delineate the effects of genetic variation in cis-regulatory genes, and the impact of haplotypes on the host immune response to malaria.