Promoter polymorphisms in the ATP binding cassette transporter gene influence production of cell-derived microparticles and are highly associated with susceptibility to severe malaria in humans.

Microparticle (MP) efflux is known to be mediated by the ABCA1 protein, and the plasma level of these cell-derived MPs is elevated considerably during human malarial infection. Therefore, two polymorphisms at positions -477 and -320 in the promoter of the ABCA1 gene were genotyped and tested for association with the plasma MP level in four groups of malaria patients segregated according to the clinical severity, i.e., cerebral malaria (CM), multiorgan dysfunction (MOD), noncerebral severe malaria, and uncomplicated malaria (UM). The TruCount tube-based flow cytometric method was used for the exact quantification of different cell-derived MPs in patients. Polymorphisms in the ABCA1 gene promoter were analyzed by use of the PCR/two-primer-pair method, followed by restriction fragment length polymorphism, in 428 malaria patients. The level of circulating plasma MPs was significantly higher in febrile patients with Plasmodium falciparum infection, especially in CM patients compared to healthy individuals. The homozygous wild-type -477 and -320 genotype was observed to be significantly higher in patients with severe malaria. These patients also showed marked increases in the plasma MP numbers compared to UM patients. We report here for the first time an association of ABCA1 promoter polymorphisms with susceptibility to severe malaria, especially to CM and MOD, indicating the protective effect of the mutant variant of the polymorphism. We hypothesize that the -477T and -320G polymorphisms affect the downregulation of MP efflux and may be a predictor of organ complication during P. falciparum malarial infections.

Endothelial nitric oxide synthase gene polymorphisms and Plasmodium falciparum infection in Indian adults.

To explore the hypothesis that susceptibility to cerebral malaria is influenced by genetic variation in endothelial nitric oxide synthase (eNOS), we genotyped three commonly defined polymorphic loci of eNOS, Glu(298)-->Asp, intron 4 variable number of tandem repeat region, and T-786-->C, in 244 patients (mean age, 36.2 years) with mild malaria and 194 patients (mean age, 35.6 years) with severe malaria belonging to same ethnic group in Orissa, an eastern Indian state. We found that there was an association of the Glu(298)-->Asp substitution (P = 0.0037; odds ratio, 1.95; 95% confidence interval, 1.2 to 3.0) and a single unique haplotype defined by "C-b-Asp" (P(corrected) = 0.0024) for protection against cerebral malaria. Further, the median plasma level of nitrite-nitrate was found to be increased in individuals with the Glu(298)-->Asp substitution and was significantly higher in the mild malaria group (P Asp substitution and the "C-b-Asp" haplotype may enhance eNOS expression and NO production, which leads to protection against cerebral malaria. These findings may increase our understanding of the pathogenesis of malaria.

Association of TNF level with production of circulating cellular microparticles during clinical manifestation of human cerebral malaria.

Microparticles (MPs) resulting from vesiculation of different cell types in Plasmodium falciparum infection correlate with the level of proinflammatory cytokine TNF that may thereby determine the disease severity. Using TruCount tube based flow cytometric method for the exact quantification of MP and enzyme linked immunosorbent assay for the measurement of TNF, we conducted a hospital based case control study on P. falciparum malaria patients to scrutinize and infer the link between the two. In 52 cerebral malaria (CM), 21 multi-organ-dysfunction (MOD), 12 non cerebral severe malaria (NCSM) and 43 uncomplicated malaria patients, the MP level was found to be significantly elevated in febrile malaria patients compared to healthy controls and a striking decrease in MP level was observed with the clearance of the P. falciparum infection in the patients upon follow-up. The lowering of the parasite density with the level of plasma TNF and the positive correlation of the cytokine with the cell derived MPs and negative correlation with the respective cell count in human malaria patients suggests that TNF may be a key stimulant to the cells resulting in the release of MPs in malaria infection.

Blood group phenotypes A and B are risk factors for cerebral malaria in Odisha, India.

There is increasing evidence that the ABO blood group phenotypes modulates Plasmodium falciparum rosetting and may influence the clinical manifestation of severe malaria. Whether blood group phenotypes are associated with risk of severe falciparum malaria in Odisha, we analyzed 343 adult malaria patients. The results showed high prevalence of blood group B in both mild (n=110) and severe malaria (cerebral malaria [CM]; n=130 and non-cerebral severe malaria [NCSM]; n=103) categories among the non-O group and while type O is significantly associated with protection against CM, patients with type A and B group had increased risk for developing CM. Further, the strength of association for B group (p=< 0.0001) was high and has double the risk of (OR=5.0) of developing CM compared to blood group A (OR=2.5). Such findings may probably be due to strain specific blood group preferences of P. falciparum and high prevalence of B group. However, the ABO blood group distribution of mild malaria was comparable with that of the NCSM group of patients. The lack of association of ABO phenotypes with NCSM is evidence for the hypothesis that the underlying pathogenesis cascades are different in CM and NCSM clinical presentations.

High CR1 level and related polymorphic variants are associated with cerebral malaria in eastern-India.

The complement receptor 1 (CR1/CD35) protein acts as the major rosetting receptor in Plasmodium falciparum infection and several genetic variants of CR1 gene have been shown to be associated with quantitative expression of erythrocyte CR1 (E-CR1) level. However, CR1 level and gene polymorphisms exhibit differences in clinical manifestation of malaria in regions of varying disease endemicity. The result of the present study which analyzed three SNPs (intron 27 HindIIIA>T, exon 22 3650 A>G, and exon 33 5507 C>G) of the CR1 gene in Orissa, a hyperendemic state in eastern-India showed that a significantly increased risk for cerebral malaria (CM) was associated with AA genotype of both intron 27 and exon 22 when compared with mild, severe malaria anemia (SMA) and CM+SMA group respectively. Further, the overall haplotype analysis for all the three loci showed predominantly two major haplotypes 'AAC' coding for higher expression of CR1 and 'TGG' haplotype coding for low expression of CR1 level with the former haplotype being significantly associated with CM (P value<0.00619 after Bonferroni correction) compared to mild malaria. The 'TGG' haplotype was proportionately more in SMA cases compared to mild malaria though statistically not significant. These findings suggest that the mild malaria group had an intermediate level of E-CR1 and extremely low or high levels of CR1 can cause severity in malaria. Further large scale studies in different endemic regions are needed to explain the epidemiological differences between E-CR1 expression and clinical manifestation of malaria which may contribute to the understanding of malaria pathogenesis.

Gene polymorphisms in angiotensin I converting enzyme (ACE I/D) and angiotensin II converting enzyme (ACE2 C-->T) protect against cerebral malaria in Indian adults.

To explore the hypothesis that angiotensin II may play a role in the susceptibility to cerebral malaria (CM), we performed a genetic association study of malaria patients in Orissa, India analyzing three SNPs (ACE2 C-->T, iNOS C-->T, eNOS Glu-->Asp) and two I/D polymorphisms (ACE I/D and IL-4 B1/B2). Our results showed that the 'D' allele of ACE I/D polymorphism, responsible for increased Ang II production had a significant association with mild malaria and the ACE2 C-->T substitution had gender specific effect of possibly reduced expression of ACE2 in presence of 'T' allele in women leading to increased level of Ang II and hence protection against CM. Combined genotype analysis of eNOS Glu-->Asp substitution responsible for increased NO production in Plasmodium falciparum infected individuals and ACE I/D polymorphism also showed stronger association of (Glu-Asp+Asp-Asp/ID+DD) genotypes with mild malaria (P<0.0001). Whether by its antiplasmodial activity and/or by some unknown mechanisms, Ang II protects from susceptibility to cerebral malaria remains to be investigated. These genetic findings may contribute to the understanding of malaria pathogenesis.

Genetic variation in neuronal nitric oxide synthase (nNOS) gene and susceptibility to cerebral malaria in Indian adults.

The role of nitric oxide (NO) in the pathogenesis of cerebral malaria is controversial. Of the three isoforms of nitric oxide synthases (NOS), though iNOS expression is the major source of NO level in vivo, nNOS is the main isoform constitutively expressed in the neural tissues. However, there has been no investigation of the role of polymorphisms of the nNOS gene in the etiology of cerebral malaria. We have analyzed two single nucleotide polymorphisms (SNPs) of nNOS gene (-84G-->A and 276C-->T), responsible for decreased basal transcriptional activity, in 200 patients with mild Plasmodium falciparum malaria and 170 patients with cerebral malaria. Our results showed a significant association of AG genotype (OR=1.83, 95%CI=1.19-2.78, P=0.007) and AA genotype (OR=3.86, 95%CI=1.42-10.5, P=0.007) of nNOS -84G-->A substitution with cerebral malaria. Interestingly, when the nNOS variant genotypes were combined together for analysis, a significantly increased risk of cerebral malaria was associated with -84(AG+AA)/276(CT+TT) genotype (OR=2.59, 95%CI=1.46-4.60, P=0.0016) and -84(AG+AA)/276(CC) genotype (OR=1.89, 95%CI=1.08-3.32, P=0.0334). The -84A seems to be a putative risk allele on the susceptibility to cerebral malaria and low nitric oxide production might have contributed to the development of cerebral malaria.