Jintana Patarapotikul

FTO polymorphisms in oceanic populations

AbstractIt has been suggested that Neels “thrifty genotype” model may account for high body weights in some Oceanic populations, which presumably arose in modern times. In European populations, common variants (rs1421085-C, rs17817449-G, and rs9939609-A) in the fat mass and obesity (FTO associated) were recently found to be associated with body mass index (BMI) or obesity. In this study, we investigated the population frequencies of these variants in six Oceanic populations (Melanesians, Micronesians, and Polynesians) and tested for an association with BMI. Unlike European populations, the Oceanic populations displayed no significant association between the FTO polymorphisms and BMI. These variants were in strong linkage disequilibrium. The population frequencies ranged between 4.2 and 30.3% in the six Oceanic populations, and were similar to those in southeast and east Asian populations. Our study of the FTO polymorphisms has generated no evidence to support the thrifty genotype hypothesis for Oceanic populations.

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.

Dynamic RNA profiling in Plasmodium falciparum synchronized blood stages exposed to lethal doses of artesunate

BackgroundTranslation of the genome sequence of Plasmodium sp. into biologically relevant information relies on high through-put genomics technology which includes transcriptome analysis. However, few studies to date have used this powerful approach to explore transcriptome alterations of P. falciparum parasites exposed to antimalarial drugs.ResultsThe rapid action of artesunate allowed us to study dynamic changes of the parasite transcriptome in synchronous parasite cultures exposed to the drug for 90 minutes and 3 hours. Developmentally regulated genes were filtered out, leaving 398 genes which presented altered transcript levels reflecting drug-exposure. Few genes related to metabolic pathways, most encoded chaperones, transporters, kinases, Zn-finger proteins, transcription activating proteins, proteins involved in proteasome degradation, in oxidative stress and in cell cycle regulation. A positive bias was observed for over-expressed genes presenting a subtelomeric location, allelic polymorphism and encoding proteins with potential export sequences, which often belonged to subtelomeric multi-gene families. This pointed to the mobilization of processes shaping the interface between the parasite and its environment. In parallel, pathways were engaged which could lead to parasite death, such as interference with purine/pyrimidine metabolism, the mitochondrial electron transport chain, proteasome-dependent protein degradation or the integrity of the food vacuole.ConclusionThe high proportion of over-expressed genes encoding proteins exported from the parasite highlight the importance of extra-parasitic compartments as fields for exploration in drug research which, to date, has mostly focused on the parasite itself rather than on its intra and extra erythrocytic environment. Further work is needed to clarify which transcriptome alterations observed reflect a specific response to overcome artesunate toxicity or more general perturbations on the path to cellular death.

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.

Chromosome size variation in the malaria parasite of rodents, Plasmodium chabaudi

Pulsed field gradient gel electrophoresis has been used to identify at least 10 large DNA fragments in the genome of the rodent malaria species Plasmodium chabaudi. The fragments range in size from approximately 650 to 5000 kb. All the fragments contain sequences homologous to a P. berghei telomere probe, suggesting that they represent intact chromosomes. Ribosomal RNA genes and P. chabaudi cDNA sequences have been mapped to specific fragments. The fragments vary in size in different cloned isolates of the parasite. In a cross between two cloned parasites differing in the sizes of chromosomes 4 and 5, independent segregation of each chromosome occurred during meiosis.