Hathairad Hananantachai

Extended Linkage Disequilibrium Surrounding the Hemoglobin E Variant Due to Malarial Selection

The hemoglobin E variant (HbE; ( beta )26Glu-->Lys) is concentrated in parts of Southeast Asia where malaria is endemic, and HbE carrier status has been shown to confer some protection against Plasmodium falciparum malaria. To examine the effect of natural selection on the pattern of linkage disequilibrium (LD) and to infer the evolutionary history of the HbE variant, we analyzed biallelic markers surrounding the HbE variant in a Thai population. Pairwise LD analysis of HbE and 43 surrounding biallelic markers revealed LD of HbE extending beyond 100 kb, whereas no LD was observed between non-HbE variants and the same markers. The inferred haplotype network suggests a single origin of the HbE variant in the Thai population. Forward-in-time computer simulations under a variety of selection models indicate that the HbE variant arose 1,240-4,440 years ago. These results support the conjecture that the HbE mutation occurred recently, and the allele frequency has increased rapidly. Our study provides another clear demonstration that a high-resolution LD map across the human genome can detect recent variants that have been subjected to positive selection.

CD36 polymorphism is associated with protection from cerebral malaria.

The human protein CD36 is a major receptor for Plasmodium falciparum-infected erythrocytes and contributes to the pathology of P. falciparum malaria. We performed variation screening of the CD36 gene and examined the possible association between CD36 polymorphisms and the severity of malaria in 475 adult Thai patients with P. falciparum malaria. Accordingly, we identified nine CD36 polymorphisms with a high-frequency (>15%) minor allele. Of these, the frequencies of the -14T-->C allele in the upstream promoter region and the -53G-->T allele in the downstream promoter region were significantly decreased in patients with cerebral malaria compared to those with mild malaria (P=.016 for -14T-->C and P=.050 for -53G-->T). The analysis of linkage disequilibrium (LD) between the nine common polymorphisms revealed that there are two blocks with strong LD in the CD36 gene and that the -14T-->C and -53G-->T polymorphisms are within the upstream block of 35 kb from the upstream promoter to exon 8. Further association testing after the second variation screening in the upstream block indicated that the in3(TG)(12) (i.e., 12 TG repeats in intron 3) allele is most strongly associated with the reduction in the risk of cerebral malaria (odds ratio 0.59; 95% confidence interval 0.40-0.87; P=.0069). We found, by reverse-transcriptase PCR amplification, that in3(TG)(12) is involved in the nonproduction of the variant CD36 transcript that lacks exons 4 and 5. Since exon 5 of the gene is known to encode the ligand-binding domain for P. falciparum-infected erythrocytes, in3(TG)(12) itself or a primary variant on the haplotype with in3(TG)(12) may be responsible for protection from cerebral malaria in Thailand. Results of the present study suggest that LD mapping has potential for detecting a disease-associated variant on the basis of haplotype blocks.

Fcγ receptor IIA and IIIB polymorphisms are associated with susceptibility to cerebral malaria

Human FcgammaRIIA and FcgammaRIIIB exhibit genetic polymorphisms, FcgammaRIIA-131H/R and FcgammaRIIIB-NA1/NA2, coding for different capacities for IgG binding and phagocytosis. Recently, FcgammaRIIA-131R was reported to be associated with protection against high-density Plasmodium falciparum infection in Kenya. Furthermore, FcgammaRIIIB-NA1/NA2 polymorphism was shown to influence FcgammaRIIA function in an allele-specific manner. In this study, we examined a possible association of FcgammaRIIA-131H/R and FcgammaRIIIB-NA1/NA2 polymorphisms with malaria severity in 107 cerebral malaria patients, 157 non-cerebral severe malaria patients, and 202 mild malaria controls living in northwest Thailand. This study reveals that, with the FcgammaRIIIB-NA2 allele, the FcgammaRIIA-131H/H genotype is associated with susceptibility to cerebral malaria (OR 1.85, 95% CI 1.14-3.01; P=0.012), although these polymorphisms are not individually involved in the disease severity. Our results suggest that FcgammaRIIA-131H/R and FcgammaRIIIB-NA1/NA2 polymorphisms have an interactive effect on host defense against malaria infection.

Significant Association of Longer Forms of CCTTT Microsatellite Repeat in the Inducible Nitric Oxide Synthase Promoter with Severe Malaria in Thailand

A CCTTT microsatellite repeat in the inducible nitric oxide synthase (iNOS) promoter was analyzed among 256 adult patients with severe Plasmodium falciparum malaria and 179 adult patients with mild malaria living in northwestern Thailand. Genotypes with longer forms of the CCTTT repeat (alleles of > or =15 repeats) were significantly associated with severe malaria (odds ratio [OR], 2.14; P=.0029, chi(2) test). More interestingly, the summed repeat number of both microsatellite alleles in an individual was found to be a significant risk factor for severe malaria (OR, 1.11; logistic regression analysis, P=.0041). The single nucleotide substitution, -954G-->C, in the iNOS promoter was rare in Thai patients with malaria. No variations were detected in the iNOS promoter region containing functional NF-kappaB elements at -5.2, -5.5, -5.8, and -6.1 kb upstream of the iNOS transcriptional start site. Thus, a CCTTT repeat in the iNOS promoter may play a key role in the pathogenesis of severe malaria.

A single-nucleotide substitution from C to T at position -1055 in the IL-13 promoter is associated with protection from severe malaria in Thailand.

We examined a possible association of single-nucleotide polymorphisms (SNPs) in the promoters of IL-3, IL-4, and IL-13 genes on the 5q31–33, IL-3 −16T>C, IL-4 −590T>C, and IL-13 −1055C>T, with severity of malaria in 361 adult malaria patients in Thailand. The IL-13 −1055T allele showed a significant association with protection from severe malaria (OR 0.51, 95% CI 0.32–0.80; P=0.0032 by the χ2 test), while allele frequencies of IL-3 −16T>C and IL-4 −590T>C were not statistically different between mild and severe malaria patients. An IL-13 −1055C>T has been reported to alter the regulation of IL-13 production. Thus, IL-13 −1055T may show resistance to severe malaria through the alteration of IL-13 production.

Significant Association of KIR2DL3-HLA-C1 Combination with Cerebral Malaria and Implications for Co-evolution of KIR and HLA

Cerebral malaria is a major, life-threatening complication of Plasmodium falciparum malaria, and has very high mortality rate. In murine malaria models, natural killer (NK) cell responses have been shown to play a crucial role in the pathogenesis of cerebral malaria. To investigate the role of NK cells in the developmental process of human cerebral malaria, we conducted a case-control study examining genotypes for killer immunoglobulin-like receptors (KIR) and their human leukocyte antigen (HLA) class I ligands in 477 malaria patients. We found that the combination of KIR2DL3 and its cognate HLA-C1 ligand was significantly associated with the development of cerebral malaria when compared with non-cerebral malaria (odds ratio 3.14, 95% confidence interval 1.52–6.48, P = 0.00079, corrected P = 0.02). In contrast, no other KIR-HLA pairs showed a significant association with cerebral malaria, suggesting that the NK cell repertoire shaped by the KIR2DL3-HLA-C1 interaction shows certain functional responses that facilitate development of cerebral malaria. Furthermore, the frequency of the KIR2DL3-HLA-C1 combination was found to be significantly lower in malaria high-endemic populations. These results suggest that natural selection has reduced the frequency of the KIR2DL3-HLA-C1 combination in malaria high-endemic populations because of the propensity of interaction between KIR2DL3 and C1 to favor development of cerebral malaria. Our findings provide one possible explanation for KIR-HLA co-evolution driven by a microbial pathogen, and its effect on the global distribution of malaria, KIR and HLA.

A Functional Single-Nucleotide Polymorphism in the CR1 Promoter Region Contributes to Protection against Cerebral Malaria

BACKGROUND Although the level of erythrocyte complement receptor type 1 (E-CR1) expression in patients with malaria has been extensively studied, whether the level of expression of E-CR1 is associated with severe malaria remains controversial. The present study examined a possible association of polymorphisms in the CR1 gene with the severity of malaria, and it evaluated the influence of the associated polymorphism on expression of E-CR1. METHODS Seventeen single-nucleotide polymorphisms in CR1 were genotyped in 477 Thai patients who had Plasmodium falciparum malaria (203 had mild malaria, 165 had noncerebral severe malaria, and 109 had cerebral malaria). The E-CR1 expression level was measured by flow cytometry in 24 healthy Thai subjects. RESULTS The T allele of the reference single-nucleotide polymorphism rs9429942 in the CR1 promoter region was strongly associated with protection against cerebral malaria (2.2% of patients with mild malaria vs. 7.8% of patients with cerebral malaria; P = .0009; Bonferroni-adjusted Pc = .0306. The E-CR1 expression level was significantly higher in individuals with the TT genotype of rs9429942 than in individuals with the TC genotype of rs9429942 (P = .0282). CONCLUSIONS We identified a CR1 promoter allele, associated with higher E-CR1 expression, that conferred protection against cerebral malaria. Previous studies have shown that the rate of clearance of immune complexes (ICs) from the circulation is related to the E-CR1 level. These results lead to the hypothesis that the clearance of ICs regulated by E-CR1 therefore plays a crucial role in the pathogenesis of cerebral malaria.

Association of the endothelial protein C receptor (PROCR) rs867186-G allele with protection from severe malaria

BackgroundCytoadhesion of Plasmodium falciparum-infected erythrocytes to endothelial cells in microvessels is a remarkable characteristic of severe malaria. The endothelial protein C receptor (EPCR), encoded by the endothelial protein C receptor gene (PROCR), has recently been identified as an endothelial receptor for specific P. falciparum erythrocyte membrane protein 1 (PfEMP1) subtypes containing domain cassettes (DCs) 8 and 13. The PROCR rs867186-G allele (serine-to-glycine substitution at position 219 of EPCR; 219Gly) has been shown to be associated with higher levels of plasma soluble EPCR (sEPCR). In this study, the association of PROCR rs867186 with severe malaria is examined in Thai population.MethodsA total of 707 Thai patients with P. falciparum malaria (341 with severe malaria and 336 with mild malaria) were genotyped for rs867186. To assess the association of PROCR rs867186 with severe malaria, three models (dominant, recessive and allelic) were evaluated. The rates of non-synonymous and synonymous substitutions were estimated for the coding sequence of the PROCR gene.ResultsThe rs867186-GG genotype was significantly associated with protection from severe malaria (P-value = 0.026; odds ratio = 0.33; 95% confidence interval = 0.12–0.90). Evolutionary analysis provided no evidence of strong positive selection acting on the PROCR gene.ConclusionThe rs867186-GG genotype showed significant association with protection from severe malaria. The present results suggest that PfEMP1–EPCR interaction, which can mediate cytoadhesion and/or reduce cytoprotective and anti-inflammatory effects, is crucial to the pathogenesis of severe malaria.

Association of ADAMTS13 polymorphism with cerebral malaria

BackgroundCerebral malaria is one of the most severe manifestations of Plasmodium falciparum malaria. The sequestration of parasitized red blood cells (PRBCs) to brain microvascular endothelium has been shown to contribute to the pathophysiology of cerebral malaria. Recent studies reported increased levels of von Willebrand factor (VWF) and reduced activity of VWF-cleaving protease, ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), in patients with cerebral malaria.MethodsAssociation of six single nucleotide polymorphisms (SNPs) of the ADAMTS13 gene with cerebral malaria was examined in 708 Thai patients with P. falciparum malaria.ResultsAmong six SNPs, the derived allele of a SNP located in intron 28, rs4962153-A, was significantly associated with protection against cerebral malaria when 115 cerebral malaria patients were compared with 367 mild malaria patients (Fishers exact P-value = 0.0057; OR = 0.27; 95% CI = 0.096-0.76). Significant association was also detected between 115 cerebral malaria and 593 non-cerebral malaria (226 non-cerebral severe malaria and 367 mild malaria) patients (Fishers exact P-value = 0.012; OR = 0.30; 95% CI = 0.11-0.83).ConclusionsExcessive adhesion of PRBCs to the platelet-decorated ultra-large VWF (ULVWF) appears to enhance the sequestration of PRBCs to cerebral microvascular endothelium. The genetic association observed in the present study implies that the regulation of platelet-decorated ULVWF strings by ADAMTS13 may play a role in the development of cerebral malaria.

Trichuris trichiura infection is associated with the multiplicity of Plasmodium falciparum infections in Thailand.

In the last decade developments in molecular biology have enabled the extensive polymorphism and complexity of Plasmodium falciparum populations within their hosts to be revealed. Super-infections or the inoculation of several genotypes during a single mosquito bite can lead to multiple concomitant infections with different P. falciparum genotypes. Several factors can influence the multiplicity of infection (MOI). For example MOI tends to increase as transmission intensity and/or the diversity of the local P. falciparum populations increases (Paul et al. 1995; Babiker et al. 1997) whereas increasing age of the host and immunity tend to reduce MOI (Ntoumi et al. 1995; Smith et al. 1999). The results of recent studies indicate that in Thailand at least interactions in humans between helminths and malaria lead to some mutual benefits not only for the parasites but also for their hosts. Some helminths appear to influence the immune response during malaria protecting against severe malaria (Nacher et al. 2000 2001b 2002a) but they also seem to increase the incidence of malaria (Nacher et al. 2002b) and to favour mixed P. falciparum/P. vivax infections (Nacher et al. 2001a). The aim of the present case-series study was to determine whether helminth infections had any influence on the multiplicity of P. falciparum infection. (excerpt)