Genetic association study of interferon lambda 3, CD27, and human leukocyte antigen-DPB1 with dengue severity in Thailand.

BACKGROUND: Dengue patients develop different disease severity ranging from mild (dengue fever [DF]) to severe forms (dengue hemorrhagic fever [DHF] and the fatal dengue shock syndrome [DSS]). Host genetics are considered to be one factor responsible for the severity of dengue outcomes. To identify genes associated with dengue severity that have not been studied yet, we performed genetic association analyses of interferon lambda 3 (IFNL3), CD27, and human leukocyte antigen-DPB1 (HLA-DPB1) genes in Thai dengue patients. METHODS: A case-control association study was performed in 877 children (age ≤ 15 years) with dengue infection (DF, n = 386; DHF, n = 416; DSS, n = 75). A candidate single nucleotide polymorphism of each of IFNL3, CD27, and HLA-DPB1 was selected to be analyzed. Genotyping was performed by TaqMan real-time PCR assay, and the association with dengue severity was examined. RESULTS: The rs9277534 variant of HLA-DPB1 was weakly associated with DHF. The genotype GG and G allele conferred protection against DHF (p = 0.04, odds ratio 0.74 for GG genotype, p = 0.03, odds ratio 0.79 for G allele). The association became borderline significant after adjusting for confounders (p = 0.05, odds ratio 0.82). No association was detected for IFNL3 or CD27. CONCLUSIONS: The present study demonstrated the weak association of the rs9277534 variant of HLA-DPB1 with protection against DHF. This variant is in the 3' untranslated region and affects HLA-DPB1 surface protein expression. Our finding suggests that HLA-DPB1 may be involved in DHF pathogenesis.

Interferon lambda 1 is associated with dengue severity in Thailand.

OBJECTIVES: Patients with dengue exhibit a range of symptoms from an acute febrile illness (dengue fever, DF), to dengue hemorrhagic fever (DHF), and to the most severe outcome, dengue shock syndrome (DSS). This study was performed to determine the host genetic factors responsible for dengue severity. Two single nucleotide polymorphisms (SNPs) of the interferon lambda 1 (IFNL1) gene (rs30461 and rs7247086) were analyzed for their association with dengue severity in a Thai population. METHODS: This was a case-control association study involving 877 patients under the age of 15 years (DF, n = 386; DHF, n = 416; DSS, n = 75). Genotyping was performed by TaqMan real-time PCR assay. RESULTS: It was found that the rs7247086 variant of IFNL1 was associated with DHF, but not DSS. Genotypes CT and TT and the T allele were protective against DHF (p = 0.03, odds ratio 0.62 for CT, odds ratio 0.13 for TT; and p = 0.01, odds ratio 0.54 for the T allele). The other SNP tested was not associated with DHF or DSS. CONCLUSIONS: The rs7247086 variant of IFNL1 (the T allele) was found to be protective against DHF, suggesting that IFNL1 may play a role in the pathogenesis of DHF.

microRNA-27a and microRNA-146a SNP in cerebral malaria.

BACKGROUND: During Plasmodium falciparum infection, microRNA expression alters in brain tissue of mice with cerebral malaria compared to noninfected controls. MicroRNA regulates gene expression post-transcriptionally to influence biological processes. Cerebral malaria pathology caused mainly by the immunological disorder. We hypothesize that single-nucleotide polymorphism in a microRNA influences microRNA biogenesis or target gene recognition and altering susceptibility to cerebral malaria. METHODS: We performed a literature search based on immunological mechanism and applied microRNA-related single-nucleotide polymorphisms database to examine candidate microRNA SNPs possibly responsible for cerebral malaria. MicroRNA-27a and microRNA-146a are supposed to involve in cerebral malaria pathology. To assess the relationship of microRNA SNP to cerebral malaria outcome, we performed TaqMan Genotyping Assays in 110 cerebral malaria and 207 uncomplicated malaria cases for three candidate microRNA SNPs (rs895819 of microRNA-27a, rs57095329 and rs2910164 of microRNA-146a). RESULTS: Our study detected no significant difference in genotype and allele frequency of individual microRNA SNPs as well as in haplotypes of microRNA-146a between these two groups of malaria patients in Thailand. Hardy-Weinberg disequilibrium of rs57095329 in the cerebral malaria group showed a heterozygous excess which might be due to natural selection. CONCLUSION: Our data supported that the candidate microRNA SNPs have no major role to develop cerebral malaria.

Correction: Sequence variation in Plasmodium falciparum merozoite surface protein-2 is associated with virulence causing severe and cerebral malaria.

[This corrects the article DOI: 10.1371/journal.pone.0190418.].

Sequence variation in Plasmodium falciparum merozoite surface protein-2 is associated with virulence causing severe and cerebral malaria.

Parasite virulence, an important factor contributing to the severity of Plasmodium falciparum infection, varies among P. falciparum strains. Relatively little is known regarding markers of virulence capable of identifying strains responsible for severe malaria. We investigated the effects of genetic variations in the P.f. merozoite surface protein 2 gene (msp2) on virulence, as it was previously postulated as a factor. We analyzed 300 msp2 sequences of single P. falciparum clone infection from patients with uncomplicated disease as well as those admitted for severe malaria with and without cerebral disease. The association of msp2 variations with disease severity was examined. We found that the N allele at codon 8 of Block 2 in the FC27-like msp2 gene was significantly associated with severe disease without cerebral complications (odds ratio = 2.73, P = 0.039), while the K allele at codon 17 of Block 4 in the 3D7-like msp2 gene was associated with cerebral malaria (odds ratio = 3.52, P = 0.024). The data suggests possible roles for the associated alleles on parasite invasion processes and immune-mediated pathogenicity. Multiplicity of infection was found to associate with severe disease without cerebral complications, but not cerebral malaria. Variations in the msp2-FC27-block 2-8N and 3D7-block 4-17K allele appear to be parasite virulence markers, and may be useful in determining the likelihood for severe and cerebral malaria. Their interactions with potential host factors for severe diseases should also be explored.

Evolution of Fseg/Cseg dimorphism in region III of the Plasmodium falciparum eba-175 gene.

The 175-kDa erythrocyte binding antigen (EBA-175) of the malaria parasite Plasmodium falciparum is important for its invasion into human erythrocytes. The primary structure of eba-175 is divided into seven regions, namely I to VII. Region III contains highly divergent dimorphic segments, termed Fseg and Cseg. The allele frequencies of segmental dimorphism within a P. falciparum population have been extensively examined; however, the molecular evolution of segmental dimorphism is not well understood. A comprehensive comparison of nucleotide sequences among 32 P. falciparum eba-175 alleles identified in our previous study, two Plasmodium reichenowi, and one P. gaboni orthologous alleles obtained from the GenBank database was conducted to uncover the origin and evolutionary processes of segmental dimorphism in P. falciparum eba-175. In the eba-175 nucleotide sequence derived from a P. reichenowi CDC strain, both Fseg and Cseg were found in region III, which implies that the original eba-175 gene had both segments, and deletions of F- and C-segments generated Cseg and Fseg alleles, respectively. We also confirmed the presence of allele with Fseg and Cseg in another P. reichenowi strain (SY57), by re-mapping short reads obtained from the GenBank database. On the other hand, the segmental sequence of eba-175 ortholog in P. gaboni was quite diverged from those of the other species, suggesting that the original eba-175 dimorphism of P. falciparum can be traced back to the stem linage of P. falciparum and P. reichenowi. Our findings suggest that Fseg and Cseg alleles are derived from a single eba-175 allele containing both segments in the ancestral population of P. falciparum and P. reichenowi, and that the allelic dimorphism of eba-175 was shaped by the independent emergence of similar dimorphic lineage in different species that has never been observed in any evolutionary mode of allelic dimorphism at other loci in malaria genomes.

Association of BAK1 single nucleotide polymorphism with a risk for dengue hemorrhagic fever.

BACKGROUND: Dengue hemorrhagic fever (DHF) is a severe life-threatening form of dengue infection. Low platelet count is one of the characteristic clinical manifestations in patients with severe dengue. However, little is known about genetic factors in the host that cause low platelet count in patients with dengue. METHODS: A previous genome-wide association study of hematological and biochemical traits identified single nucleotide polymorphisms (SNPs) associated with low platelet count in healthy subjects. To examine the possible association of these SNPs with DHF, 918 Thai patients with dengue [509 patients with DHF and 409 with dengue fever (DF)] were genotyped for five SNPs: rs5745568 in BAK1, rs6141 in THPO, rs6065 in GP1BA, rs739496 in SH2B3, and rs385893 in RCL1. In addition, rs4804803 in CD209, that has been reported to be associated with dengue infection, was also genotyped to examine if rs4804803 affects the association detected in this study. RESULTS: The allele frequencies of each SNP were compared between the DHF and DF groups. Among the five SNPs, the G allele of rs5745568 in BAK1 was significantly associated with a risk for DHF [P = 0.006 and crude odd ratio (95 % confidence interval) = 1.32 (1.09-1.60)]. The association of this allele with DHF was also significant in a logistic regression analysis adjusted for age, sex, hospital (i.e., geographic region), immune status (i.e., primary or secondary infection), and virus serotype [P = 0.016 and adjusted odd ratio (95 % confidence interval) = 1.29 (1.05-1.58)]. The result was not influenced by rs4804803 [P = 0.0167 and adjusted OR (95 % CI) = 1.29 (1.05-1.58)]. No other SNPs including rs4804803 showed significant association. CONCLUSIONS: The low-level constitutive production of platelets caused by the G allele of rs5745568 seems to increase the risk of bleeding in dengue infection. Our results suggest that BCL-2 homologous antagonist/killer (BAK) protein, encoded by BAK1, plays a crucial role in the pathogenesis of DHF.

Association of PECAM1/CD31 polymorphisms with cerebral malaria.

Platelet/endothelial cell adhesion molecule-1 (PECAM1/CD31), a receptor recognized by P. falciparum-infected red blood cells (iRBCs), on the vascular endothelium has been implicated in mediating cytoadherence in patients with P. falciparum malaria. To examine associations of PECAM1 polymorphisms with cerebral malaria, 11 tag single nucleotide polymorphisms (SNPs) of PECAM1 were analysed for 312 Thai patients with P. falciparum malaria (109 with cerebral malaria and 203 with mild malaria). The rs1122800-C allele was significantly associated with protection from cerebral malaria (P = 0.017), and the rs9912957-A significantly increased the risk for cerebral malaria (P = 0.0065) in malaria patients. Fine-scale mapping using genotyped and imputed SNPs and linkage disequilibrium (LD) analysis revealed that rs1122800 and rs9912957 were located in two distinct LD blocks and were independently associated with cerebral malaria. The rs1122800-C allele was significantly associated with lower expression level of PECAM1 in EBV-transformed lymphoblastoid cell lines (P = 0.045). The present results suggest that PECAM1-mediated cytoadherence of iRBCs to brain endothelium plays a crucial role in the pathogenesis of cerebral malaria.

Genetic evidence for contribution of human dispersal to the genetic diversity of EBA-175 in Plasmodium falciparum.

BACKGROUND: The 175-kDa erythrocyte binding antigen (EBA-175) of Plasmodium falciparum plays a crucial role in merozoite invasion into human erythrocytes. EBA-175 is believed to have been under diversifying selection; however, there have been no studies investigating the effect of dispersal of humans out of Africa on the genetic variation of EBA-175 in P. falciparum. METHODS: The PCR-direct sequencing was performed for a part of the eba-175 gene (regions II and III) using DNA samples obtained from Thai patients infected with P. falciparum. The divergence times for the P. falciparum eba-175 alleles were estimated assuming that P. falciparum/Plasmodium reichenowi divergence occurred 6 million years ago (MYA). To examine the possibility of diversifying selection, nonsynonymous and synonymous substitution rates for Plasmodium species were also estimated. RESULTS: A total of 32 eba-175 alleles were identified from 131 Thai P. falciparum isolates. Their estimated divergence time was 0.13-0.14 MYA, before the exodus of humans from Africa. A phylogenetic tree for a large sequence dataset of P. falciparum eba-175 alleles from across the world showed the presence of a basal Asian-specific cluster for all P. falciparum sequences. A markedly more nonsynonymous substitutions than synonymous substitutions in region II in P. falciparum was also detected, but not within Plasmodium species parasitizing African apes, suggesting that diversifying selection has acted specifically on P. falciparum eba-175. CONCLUSIONS: Plasmodium falciparum eba-175 genetic diversity appeared to increase following the exodus of Asian ancestors from Africa. Diversifying selection may have played an important role in the diversification of eba-175 allelic lineages. The present results suggest that the dispersals of humans out of Africa influenced significantly the molecular evolution of P. falciparum EBA-175.

Lack of association between BSG polymorphisms and cerebral malaria.

The Ok(a) blood group antigen basigin (BSG or CD147) is an erythrocyte receptor for the PfRh5 protein from Plasmodium falciparum. A recent study has shown that the PfRh5-BSG interaction is essential for erythrocyte invasion by P. falciparum. In this study, 6 SNPs in the BSG gene were investigated in 312 adult patients with P. falciparum malaria (109 cerebral malaria and 203 mild malaria patients) living in northwest Thailand. To examine the association between BSG SNPs and cerebral malaria, the allele and haplotype frequencies were compared in cerebral and mild malaria patients. Nonsynonymous SNPs were not assessed in the association analysis. The results showed that common BSG polymorphisms and haplotypes were not significantly associated with cerebral malaria. In conclusion, common SNPs in BSG do not influence the risk of cerebral malaria in the Thai population.

A replication study confirms the association of GWAS-identified SNPs at MICB and PLCE1 in Thai patients with dengue shock syndrome.

BACKGROUND: Dengue shock syndrome (DSS), a severe life-threatening form of dengue infection, mostly occurs in children. A recent genome wide association study (GWAS) identified two SNPs, rs3132468 of major histocompatibility complex class I polypeptide-related sequence B (MICB) and rs3765524 of phospholipase C, epsilon 1 (PLCE1), associated with DSS in Vietnamese children. In this study, to examine whether an identical association is found in a different population, the association of these two SNPs with DSS was assessed in Thai children with dengue. METHODS: The rs3132468 and rs3765524 SNPs were genotyped in 917 Thai children with dengue: 76 patients with DSS and 841 patients with non-DSS. The allele frequencies were compared between DSS and non-DSS groups by one-sided Fisher's exact test. The association of rs3132468 and rs3765524 with the mRNA expression levels of MICB and PLCE1 were assessed in EBV-transformed lymphoblastoid cell lines. RESULTS: The reported DSS-risk alleles were significantly associated with DSS in Thai patients with dengue (one-sided P = 0.0213 and odds ratio [OR] = 1.58 for rs3132468-C and one-sided P = 0.0252 and OR = 1.49 for rs3765524-C). The rs3132468-C allele showed a significant association with lower mRNA level of MICB (P = 0.0267), whereas the rs3765524-C allele did not. These results imply that the MICB molecule may play an important role in the prevention of DSS in dengue infection. CONCLUSIONS: Together with previous association studies, we conclude that rs3132468-C at MICB and rs3765524-C at PLCE1 confer risk of DSS in Southeast Asians.

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

BACKGROUND: Cytoadhesion 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. METHODS: A 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. RESULTS: The 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. CONCLUSION: The 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.

Diversifying selection on the thrombospondin-related adhesive protein (TRAP) gene of Plasmodium falciparum in Thailand.

Sporozoites of Plasmodium falciparum are transmitted to human hosts by Anopheles mosquitoes. Thrombospondin-related adhesive protein (TRAP) is expressed in sporozoites and plays a crucial role in sporozoite gliding and invasion of human hepatocytes. A previous study showed that the TRAP gene has been subjected to balancing selection in the Gambian P. falciparum population. To further study the molecular evolution of the TRAP gene in Plasmodium falciparum, we investigated TRAP polymorphisms in P. falciparum isolates from Suan Phueng District in Ratchaburi Province, Thailand. The analysis of the entire TRAP coding sequences in 32 isolates identified a total of 39 single nucleotide polymorphisms (SNPs), which comprised 37 nonsynonymous and two synonymous SNPs. McDonald-Kreitman test showed that the ratio of the number of nonsynonymous to synonymous polymorphic sites within P. falciparum was significantly higher than that of the number of nonsynonymous to synonymous fixed sites between P. falciparum and P. reichenowi. Furthermore, the rate of nonsynonymous substitution was significantly higher than that of synonymous substitution within Thai P. falciparum. These results indicate that the TRAP gene has been subject to diversifying selection in the Thai P. falciparum population as well as the Gambian P. falciparum population. Comparison of our P. falciparum isolates with those from another region of Thailand (Tak province, Thailand) revealed that TRAP was highly differentiated between geographically close regions. This rapid diversification seems to reflect strong recent positive selection on TRAP. Our results suggest that the TRAP molecule is a major target of the human immune response to pre-erythrocytic stages of P. falciparum.

Association of IL1B -31C/T and IL1RA variable number of an 86-bp tandem repeat with dengue shock syndrome in Thailand.

BACKGROUND: Dengue patients present a range of symptoms: dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). It is not clear whether this variability is due to their genetic background. Here we tested polymorphisms of interleukin 1 beta (IL1B) and interleukin 1 receptor antagonist (IL1RA) genes for association with DSS in the Thai population. METHODS: Polymorphisms of IL1B -31C/T (rs1143627) and IL1RA 86-base-pair tandem repeat were analyzed in 871 patients (DF = 384, DHF = 413, and DSS = 74). RESULTS: IL1B -31C and IL1RA 2/4 genotype were associated with DSS (IL1B -31C: DSS vs DHF: P = .0061, odds ratio [OR, 95% confidence interval {CI}], 3.49 [1.36-8.95]; DSS vs DF: P = .027, OR [95% CI], 2.81 [1.12-7.06]; IL1RA 2/4: DSS vs DHF: P = .017, OR [95% CI], 1.94 [1.12-3.40]; DSS vs DF: P = .024, OR [95% CI], 1.90 [1.07-3.4]). No difference was found between DF and DHF. Logistic regression analysis revealed that IL1B -31C and IL1RA 2/4 genotypes were each independently associated with DSS. CONCLUSIONS: Patients with IL1B -31C carrier, or IL1RA 2/4 genotype carry a risk for DSS, implying that IL1B may play a role in pathogenesis of DSS.

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.

Association of ADAMTS13 polymorphism with cerebral malaria.

BACKGROUND: Cerebral 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. METHODS: Association of six single nucleotide polymorphisms (SNPs) of the ADAMTS13 gene with cerebral malaria was examined in 708 Thai patients with P. falciparum malaria. RESULTS: Among 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 (Fisher's 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 (Fisher's exact P-value = 0.012; OR = 0.30; 95% CI = 0.11-0.83). CONCLUSIONS: Excessive 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.

A replication study of the association between the IL12B promoter allele CTCTAA and susceptibility to cerebral malaria in Thai population.

BACKGROUND: Interleukin-12 (IL-12), a heterodimeric cytokine composed of p35 and p40 subunits, has been thought to play an important role in the pathogenesis of malaria. The IL-12p40 subunit is encoded by the IL12B gene. An IL12B promoter allele, CTCTAA, at rs17860508 has been reported to be associated with susceptibility to cerebral malaria in African populations. However, this association has not so far been replicated in non-African populations. METHODS: To examine whether the CTCTAA allele is associated with susceptibility to cerebral malaria in Asian populations, 303 Thai patients with Plasmodium falciparum malaria (109 cerebral malaria and 194 mild malaria patients) were genotyped for rs17860508 by PCR-direct sequencing. RESULTS: The CTCTAA allele showed a significant association with susceptibility to cerebral malaria in the Thai population (allelic OR = 1.37; one sided P-value = 0.030). CONCLUSIONS: The existence of a significant association between the CTCTAA allele and susceptibility to cerebral malaria was confirmed in Southeast Asian population, which was previously reported in African populations.

Population diversity and antibody selective pressure to Plasmodium falciparum MSP1 block2 locus in an African malaria-endemic setting.

BACKGROUND: Genetic evidence for diversifying selection identified the Merozoite Surface Protein1 block2 (PfMSP1 block2) as a putative target of protective immunity against Plasmodium falciparum. The locus displays three family types and one recombinant type, each with multiple allelic forms differing by single nucleotide polymorphism as well as sequence, copy number and arrangement variation of three amino acid repeats. The family-specific antibody responses observed in endemic settings support immune selection operating at the family level. However, the factors contributing to the large intra-family allelic diversity remain unclear. To address this question, population allelic polymorphism and sequence variant-specific antibody responses were studied in a single Senegalese rural community where malaria transmission is intense and perennial. RESULTS: Family distribution showed no significant temporal fluctuation over the 10 y period surveyed. Sequencing of 358 PCR fragments identified 126 distinct alleles, including numerous novel alleles in each family and multiple novel alleles of recombinant types. The parasite population consisted in a large number of low frequency alleles, alongside one high-frequency and three intermediate frequency alleles. Population diversity tests supported positive selection at the family level, but showed no significant departure from neutrality when considering intra-family allelic sequence diversity and all families combined. Seroprevalence, analysed using biotinylated peptides displaying numerous sequence variants, was moderate and increased with age. Reactivity profiles were individual-specific, mapped to the family-specific flanking regions and to repeat sequences shared by numerous allelic forms within a family type. Seroreactivity to K1-, Mad20- and R033 families correlated with the relative family genotype distribution within the village. Antibody specificity remained unchanged with cumulated exposure to an increasingly large number of alleles. CONCLUSION: The Pfmsp1 block2 locus presents a very large population sequence diversity. The lack of stable acquisition of novel antibody specificities despite exposure to novel allelic forms is reminiscent of clonal imprinting. The locus appears under antibody-mediated diversifying selection in a variable environment that maintains a balance between the various family types without selecting for sequence variant allelic forms. There is no evidence of positive selection for intra-family sequence diversity, consistent with the observed characteristics of the antibody response.

Identification of a haplotype block in the 5q31 cytokine gene cluster associated with the susceptibility to severe malaria.

BACKGROUND: It has been previously demonstrated that a single nucleotide polymorphism (SNP) in the IL13 promoter region, IL13 -1055T>C (rs1800925), was associated with susceptibility to severe malaria in Thais. In the present study, fine association mapping for a cytokine gene cluster including IL4, IL5, and IL13 on chromosome 5q31 was conducted using the same malaria subjects to refine the region containing a primary variant or a haplotype susceptible to severe malaria. METHODS: A total of 82 SNPs spanning 522 kb of the 5q31 region were analysed in 368 patients with Plasmodium falciparum malaria (203 mild malaria and 165 severe malaria patients). RESULTS: Only rs1881457 located in the promoter region of IL13, which is in linkage disequilibrium with rs1800925 (r2 = 0.73), showed a significant association with severe malaria after adjusting for multiple testing (P = 0.046 by permutation test). This SNP was in a haplotype block spanning 97 kb (from rs2069812 to rs2240032). The detected haplotype block contained the RAD50 gene and the promoter of IL13, but not the other genes. CONCLUSION: A haplotype block in which a primary polymorphism associated with severe malaria is likely to be encoded was identified in Thai malaria patients.

IFNGR1 polymorphisms in Thai malaria patients.

Interferon-gamma (IFN-gamma) has been suggested to play an important role in the pathogenesis of malaria. To examine possible association of the IFN-gamma receptor 1 (IFNGR1) polymorphisms with cerebral malaria, 312 adult patients with Plasmodium falciparum malaria (203 mild and 109 cerebral malaria patients) living in northwest Thailand were genotyped for six single nucleotide polymorphisms (SNPs) including -56T/C (rs2234711) and a microsatellite marker in IFNGR1. A case-control association analysis failed to detect significant association between the IFNGR1 polymorphisms and cerebral malaria, thus implying that the IFNGR1 polymorphism may not be a major genetic factor influencing the development of cerebral malaria in the Thai population. These data also provide useful information for future genetic studies of IFNG polymorphisms in Thai patients.