The TLR9 polymorphisms have been postulated to have a cis-regulatory effect on TLR9 expression  and also shown to alter cytokine levels during severe malaria infections . In this study, the effect of TLR9 gene polymorphisms on symptomatic malaria was investigated and TLR9 polymorphisms and haplotypes were significantly associated with susceptibility to symptomatic malaria among Ghanaian children.
In this cohort study, the intronic polymorphism rs352139 (G + 1174A) GG genotype was significantly associated with increased risk for symptomatic malaria and high parasitaemia. Previous functional study has reported that this intronic SNP has a regulatory effect on TLR9 expression, with the rs352139 G allele in combination with the promoter rs187084 (C-1486 T) C allele having a down-regulatory effect, while the rs352139 A allele in combination with rs187084 T allele having an up-regulatory effect on TLR9 expression among patients with systemic lupus erythematosus . However, it is currently not clear how this intronic SNP induces such a phenotype change. It is possible that it influences signalling by creating an alternative splicing site and thus, affecting the mRNA transcription and the final protein product. Equally, the rs352139 SNP could be a likely marker in LD with a polymorphic regulatory region that controls TLR9 expression or a functional coding region SNP. In contrast to our finding, Campino et al reported an association of the rs352139 A allele with severe malaria among Malawian population but not in the Gambian population . This discrepancy may in part be explained by the difference in phenotypes and populations examined, and also by the different role pro-inflammatory cytokines play in the pathogenesis of symptomatic and severe malaria. For example, an early high IFN-γ response has been reported to confer protection against symptomatic malaria episodes [31, 32], while an over-production of IFN-γ has been associated with susceptibility to cerebral malaria .
The synonymous coding SNP, rs352140 (G + 2848A) GG genotype was associated with protection from symptomatic malaria but no such protective effect was observed for parasitaemia among our cohort population. This rs352140 SNP is linked to the rs352139 (G + 1174A) (D1 = 1, r2 =0.237) in the study population, with the rs352140 G allele linked to rs352139 A allele. Thus, the observed protective effect could partly be attributed to the effect of the rs352139 A allele which has been shown to have an up-regulatory effect on TLR9 expression .
The promoter polymorphism rs5743836 (C-1237 T) TT genotype was associated with low parasitaemia but no effect on susceptibility to symptomatic malaria was observed in this study. Our finding is consistent with that of Leorrati et al who reported an association of rs5743836 TT genotype with low parasitaemia (<10000 parasite/μl) among adults with mild malaria in the Brazilian Amazon . The rs5743836 TT variant has been shown to have a higher promoter activity than the CC genotype , and thus, could result in increased pro-inflammatory cytokine production during malaria infection leading to successful control and elimination of malaria parasites.
On haplotype analysis, the TLR9 TTAG (−1486 T, -1237 T, +1174A, +2848 G) haplotype, the most frequent haplotype (39.1%) in the study population, was significantly associated with protection against symptomatic malaria and high parasitaemia. These findings were strongly supported by the result of luciferase reporter gene expression assay which showed a significantly higher promoter activity for the TTA (−1486 T, -1237 T, +1174A) haplotype compared with the CCG, CTG and TCG haplotypes (Figure 1b). It was also observed that the CTG haplotype had a higher promoter activity than the CCG haplotype (Figure 1b). Taken together, the haplotype with A allele for rs352139 (TTA) consistently had a significantly higher promoter effect than all the haplotypes with G allele for the same SNP (i.e. CCG, CTG and TCG). It was also observed that the haplotypes with T allele for rs5743836 (i.e. TTA, CTG) had a higher promoter effect than those with C allele for the same SNP (i.e. CCG, TCG) and that carriers of the TTA haplotype had the highest promoter activity, the CTG haplotype had an intermediate activity, and the CCG having the lowest promoter activity. Thus, it is possible that these two SNPs (the rs5743836 (C-1237 T) and rs352139 (G + 1174A)) have a greater role of influencing TLR9 gene transcription and expression. This luciferase assay result is in agreement with the findings of Tao et al 2007, who reported that the rs187084 (C-1486 T) T allele in combination with rs352139 (G + 1174A) A allele had a higher TLR9 expression compared with the rs187084 C allele in combination with rs352139 G allele among Japanese population who are not polymorphic for rs5743836 (C-1237 T) (i.e. TTA had higher TLR9 expression than CTG) (31). Therefore, it can be inferred that the TTAG haplotype may exert an increased TLR9 transcriptional activity resulting in higher TLR9 expression and subsequently higher pro-inflammatory cytokine production, thus conferring protection against symptomatic malaria phenotype. The TTAG haplotype has also been associated with increased risk for meningococcal meningitis among Dutch children  and also associated with protection from ulcerative colitis among the Japanese population . The different effects of TLR9 haplotypes in different disease phenotypes could be explained by the different roles inflammatory cytokines play in pathogenesis of these diseases. While robust pro-inflammatory cytokines has been reported to provide protection against clinical mild malaria  and against experimental colitis in mice ; in bacterial meningitis robust inflammatory response has been identified as harmful and contributing to tissue damage .
The major limitation of this study is the low incidence of symptomatic malaria . The study was conducted in an area with low but perennial malaria transmission with the intention of identifying clearly individuals susceptible or resistant to the symptomatic malaria phenotypes. Nevertheless, this study has highlighted clearly that the TLR9 gene polymorphisms significantly influence susceptibility to symptomatic malaria. It is therefore worthwhile to carry out more studies in areas with different malaria transmission and using a larger sample size, so that the role of TLR9 polymorphisms on different malaria phenotypes can clearly be deciphered.