SEARCH

SEARCH BY CITATION

Keywords:

  • cerebral malaria;
  • TIM1;
  • promoter;
  • polymorphism;
  • Thailand

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Although cerebral malaria is a major life-threatening complication of Plasmodium falciparum infection, its pathophysiology is not well understood. Prolonged activation of the T helper type 1 (Th1) response characterized by the production of pro-inflammatory cytokines such as IFN-γ and TNF-α has been suggested to be responsible for immunopathological process leading to cerebral malaria unless they are downregulated by the anti-inflamatory cytokines produced by the Th2 response. The T cell immunoglobulin and mucin domain (TIM) family of proteins are cell surface proteins involved in regulating Th1 and Th2 immune responses. In this study, the possible association between the polymorphisms of TIM1, TIM3, and TIMD4 genes and the severity of malaria was examined in 478 adult Thai patients infected with P. falciparum malaria. The TIM1 promoter haplotype comprising three derived alleles, -1637A (rs7702919), -1549C (rs41297577) and -1454A (rs41297579), which were in complete linkage disequilibrium, was significantly associated with protection against cerebral malaria (OR = 0.41; 95% CI = 0.24–0.71; P= 0.0009). Allele-specific transcription quantification analysis revealed that the level of mRNA transcribed from TIM1 was higher for the protective promoter haplotype than for the other promoter haplotype (P= 0.004). Engagement with TIM1 in combination with T cell receptor stimulation induces anti-inflammatory Th2 cytokine production, which can protect the development of cerebral malaria caused by overproduction of pro-inflammatory Th1 cytokines. The present results suggest that the higher TIM1 expression associated with the protective TIM1 promoter haplotype confers protection against cerebral malaria.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Malaria is a debilitating infectious disease that is commonly found in tropical and sub-tropical countries. In any given year, 300–500 million cases of malaria are reported, and at least 1 million people die. There are four human malaria parasites, of which Plasmodium falciparum is responsible for cerebral malaria which is a major cause of death due to malaria. Although the pathogenesis of cerebral malaria is not well understood, host genetic factors are thought to influence the clinical outcome (Hill 1999).

During the acute stage of malaria infection, the T helper type 1 (Th1) immune response, characterized by the production of pro-inflammatory cytokines such as IFN-γ and TNF-α, plays an important role in the elimination of parasites. In addition, these Th1 cytokines are thought to be involved in the development of cerebral malaria (Hunt & Grau 2003). Production of Th1 cytokines can be inhibited by Th2 cytokines, so it is possible that relative overproduction of Th1 cytokines and/or underproduction of Th2 cytokines could lead to the development of cerebral malaria (Hunt & Grau 2003). Therefore, polymorphisms of genes involved in the balance between Th1 and Th2 responses may potentially be important host factors contributing to development of cerebral malaria.

The T cell immunoglobulin and mucin (TIM) gene family encodes cell surface proteins involved in the regulation of the Th1 and Th2 immune responses. The human TIM gene family consists of three members, the genes encoding TIM1 (OMIM 606518), TIM3 (OMIM 606652), and T-cell immunoglobulin and mucin domain containing protein 4 (TIMD4; OMIM 610096), all of which are located on chromosome 5q33.2 (Figure 1A). The TIM1 gene, which was initially cloned as kidney injury molecule 1 (KIM1) and hepatitis A virus cellular receptor 1 (HAVCR1), encodes a type 1 transmembrane glycoprotein composed of an immunoglobulin variable region (IgV)-like domain, a mucin-like domain, a transmembrane region and a cytoplasmic tail (Feigelstock et al. 1998a, 1998b; Han et al. 2002; Ichimura et al. 1998; Kaplan et al. 1996). The TIM3 gene encodes a 301 amino-acid type I transmembrane glycoprotein. TIM3 is preferentially expressed on Th1 cells in humans and mice (Khademi et al. 2004; Monney et al. 2002). Although the natural ligand of TIM3 has not been identified, TIM3 functions as a specific down-regulator of Th1-type immune response in mice (Sabatos et al. 2003; Sanchez-Fueyo et al. 2003). The mouse TIMD4 gene encodes a protein that is the natural ligand of TIM1 and is expressed on antigen-presenting cells, especially mature lymphoid dendritic cells (DCs) (Meyers et al. 2005). The interaction between TIMD4 on the surface of DCs and TIM1 on the surface of activated T cells delivers a stimulatory signal into the T cell and leads to the enhancement of T cell expansion and effector functions (Meyers et al. 2005).

image

Figure 1. (A) Genomic structure of the TIM gene family, members of which are located on human chromosome 5q33.2. The physical position is as shown in the NCBI human genome build 35.1, released on Jan 6, 2005. (B) P-values for the detected polymorphisms in the TIM gene region. The P-values were obtained from a chi-squared association test, in which the allele frequencies were compared for 207 patients with mild malaria and 108 patients with cerebral malaria. (C) Pairwise D′ values for 207 Thai patients with mild malaria. Bright red squares indicate high D′ values (D′= 1) and high LOD scores (LOD > = 2), and light blue squares indicate high D′ values (D′= 1) and low LOD scores (LOD < 2). For other cases, the D′ value is shown in each square. It should be noted that TIM1 g.15251 is not included here since it was monomorphic in mild and cerebral malaria patients. Red and blue asterisks indicate polymorphic markers with a significant P-value (P < 0.05) in the TIM1 promoter region and in the TIM1 intragenic region, respectively.

Download figure to PowerPoint

In this study, we screened for polymorphisms of TIM1, TIM3 and TIMD4 in Thai patients infected with P. falciparum, and found a statistically significant association between protection against cerebral malaria and the TIM1 promoter haplotype. We further observed that TIM1 with the protective promoter haplotype was transcribed at a higher level relative to the other promoter haplotype.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Malaria Patients

A total of 478 Thai patients with P. falciparum malaria (207 with mild malaria, 163 with non-cerebral severe malaria and 108 with cerebral malaria), living in northwest Thailand near the border with Myanmar, were enrolled in this study. All patients underwent treatment at the Hospital for Tropical Disease, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. Clinical manifestations of malaria were classified according to the definitions and associated criteria by the World Health Organization. Cerebral malaria was defined as unrousable coma, a positive result in tests for the presence of an asexual form of P. falciparum, and exclusion of other causes of coma. Mild malaria was defined as having a positive blood smear and fever without other causes of infection and no signs indicating severe malaria as described below. Non-cerebral severe malaria was defined as above but with the presence of one of the following signs: high parasitemia (>100,000 parasites/ml), hypoglycemia (glucose level < 22 nmol/L), severe anemia (hematocrit < 20% or hemoglobin level < 7.0 g/dl) or increased serum level of creatinine (>3.0 mg/dl). The individuals recruited in this study were aged 13 years or more. The average ages for patients with mild, non-cerebral severe, and cerebral malaria were 26.0, 24.5 and 28.6 years, respectively. Genomic DNA was extracted from peripheral blood leukocytes by using the QIAmp Blood Kit (Qiagen, Hilden, Germany). This study was approved by the institutional review board of the Faculty of Tropical Medicine, Mahidol University, and the Research Ethics Committee of the Faculty of Medicine, The University of Tokyo. Informed consent was obtained from all patients.

Variation Screening and Genotyping of the TIM Gene Family

Variation screening of (i) all exons with neighboring introns and (ii) an approximately 2 kb putative promoter region located upstream of the translation initiation sites of the TIM1, TIM3, and TIMD4 genes was performed by PCR direct sequencing using specific primer sets designed on the basis of the nucleotide sequence NC_000005.8. In the variation screening, 48 malaria patients (16 with mild malaria, 16 with non-cerebral severe malaria and 16 with cerebral malaria) were analyzed for the detection of polymorphic sites. The PCR reactions were carried out using Applied Biosystem reagents and AmpliTaq Gold DNA polymerase (Perkin Elmer, Norwalk, CT, USA). PCR was carried out using a BioMetra PCR machine with the following conditions: initial denaturation at 96˚C for 10 min; followed by 40 cycles of 96˚C for 30 s, annealing temperature for 45 s, and 72˚C for 30 s; then finally 72˚C for 7 min. Fluorescence-based cycle labeling of PCR products was performed using the BigDye® Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA). The labeled products were subjected to analysis using the ABI Prism 3100 Genetic Analyzer (Applied Biosystems) in accordance with the manufacturer's instructions. The detected polymorphisms were analyzed further by using PCR-direct sequencing, PCR-single strand conformation polymorphism (SSCP) analysis or PCR-restriction fragment length polymorphism (RFLP) analysis. The protocols for PCR-SSCP and PCR-RFLP are available upon request from the authors. The PCR primers and annealing temperatures used for genotyping the identified polymorphisms are listed in supplementary Table 1.

To investigate the allele and haplotype frequencies of TIM1 promoter polymorphisms in other populations, TIM1 -1637G>A, TIM1 -1549G>C, and TIM1 -1454G>A were also analyzed in two populations studied in the HapMap project (The International HapMap Consortium. 2003): 58 Yoruba in Ibadan, Nigeria (YRI), and 45 Han Chinese in Beijing, China (CHB).

Allele-Specific Transcript Quantification

A total of 96 unrelated healthy Thai individuals underwent allele-specific transcript quantification analysis. All subjects were at least 20 years old, with an average age of 24 years. Genomic DNA and total RNA were extracted from peripheral blood leukocytes by using the QIAmp Blood mini Kit (Qiagen) and the PAXgene Blood RNA system (PreAnalytiX, Hombrechtikon, Switzerland) in accordance with the respective manufacturers' instructions. To avoid contamination with genomic DNA, the DNA-free™ DNase Treatment and Removal Reagent (Ambion, USA) was used in accordance with the manufacturer's instructions. That genomic DNA had been successfully removed from the RNA samples was verified by failure to amplify the TIM 1 gene using PCR.

To compare the transcription levels of the H1 and H2 TIM1 promoter haplotypes (H1 comprises -1637G, -1549G and -1454 G, and H2 comprises -1637A, -1549C and -1454 A), we used a coding polymorphism in exon 4 (g.5383_5397indel) as a tag for measuring the proportion of allele-specific transcripts in g.5383_5397indel heterozygous individuals. To determine the haplotype phase for promoter single nucleotide polymorphisms (SNPs) (-1637 G>A, -1549 G>C and -1454 G>A) and the g.5383_5397indel, allele-specific long-range PCR amplification was performed by using an allele-specific reverse primer for g.5383_5397Ins and a forward primer located in the promoter. The PCR products were analyzed by PCR-direct sequencing.

RNA samples were subjected to reverse transcription using PrimeScript One Step RT-PCR (TaKaRa Bio Inc., Japan). The primers used for RT-PCR were TIM1_ex3F and TIM1_ex7R (supplementary Table 2). Then, the RT-PCR products were subjected to second PCR amplification using TIM1_ex4F and TIM1_ex4R (supplementary Table 2). The PCR products, which had different lengths because of the g.5383_5397indel (278 bp for Del and 293 bp for Ins), were analyzed by capillary electrophoresis using the Agilent 2100 BioAnalyzer (Agilent Technology, CA, USA) and the DNA 500 LabChip Kit (Agilent Technology). The ratio of fluorescence intensity for the two RT-PCR products distinguished by the g.5383_5397indel was obtained for two diplotypes (six H2-Del/H1-Ins individuals and six H1-Del/H1-Ins individuals). The ratio of fluorescence intensity for two PCR products (i.e., Del/Ins) derived from the genomic DNA of the same subject was also analyzed to evaluate the variance. In this study, the allelic expression ratio of g.5383_5397Del to g.5383_5397Ins was obtained by dividing the individual ratio for the RT-PCR products by the mean of the ratio for the PCR products for the genomic DNA. All experiments were carried out in triplicate for each subject. The allele-specific transcript quantification analysis used in this study has the advantage that the natural transcription machinery is operating in the native chromatin environment. It is therefore a powerful in vivo approach for detecting the cis-acting effect of polymorphisms on gene expression (Kimura et al. 2004, 2005).

Statistical Analysis

The chi-squared test and Fisher's exact test were used for comparing the genotype and allele frequencies of TIM gene polymorphisms between two groups of Thai patients with malaria. It should be noted here that all the polymorphisms were tested for association with the severity of malaria regardless of the deviation from Hardy-Weinberg equilibrium in each malaria group because the deviation may have come from the significant effect of the tested polymorphism on protection against severe malaria or on susceptibility to severe malaria. To evaluate the extent of linkage disequilibrium (LD) between polymorphisms, we calculated pairwise LD coefficients (D′) and visualized them using the Haploview program (Barrett et al. 2005). The frequencies of the promoter haplotypes comprising -1637 G>A, -1549 G>C, and -1454 G>A were estimated using Haploview (Barrett et al. 2005). The Mann-Whitney U-test was used to examine the allelic expression imbalance or the difference in allelic expression ratios between two diplotypes (H2-Del/H1-Ins v.s. H1-Del/H1-Ins). A P-value less than 0.05 was considered to be statistically significant in this study.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Polymorphisms in TIM Genes

The polymorphisms detected in this study are shown in Table 1. In TIM1 and TIMD4, 16 and three polymorphisms were identified, respectively, while no polymorphism was found in TIM3. Polymorphisms causing amino acid changes were observed only in exon 4 of TIM1. To determine which were the ancestral and which the derived alleles, human TIM1 and TIMD4 sequences were compared with the chimpanzee genome (Pan troglodytes) using the BLAST tool of the NCBI database. The allele found in chimpanzee was inferred to be the ancestral allele, and the other was regarded as the derived allele (Table 1).

Table 1.  Polymorphisms detected in Thai patients with malaria
dbSNP NumberChromosomal coordinateaPolymorphism name with position relative to the first ATG codonbAlleles (ancestral/derived)Region
  1. aThe coordinate on chromosome 5 is as shown in the NCBI human genome build 35.1, released on Jan 6, 2005.

  2. bThe positions of the described sequence variants were determined based on the NCBI reference sequence (NC_000005.8). The nucleotide numbering system uses the A of the ATG translation initiation sequence as nucleotide +1. Promoter, exon and exon-intron boundaries are retrieved from the UCSC Genome Browser on Human Assembly released on May 2004.

rs2116787156432559−15127T/CIntergenic
rs4704844156427179− 9747T/CIntergenic
rs7702919156419169TIM1 -1637G/APromoter
rs41297575156419101TIM1 -1569G/APromoter
rs41297577156419081TIM1 -1549G/CPromoter
rs41297579156418986TIM1 -1454G/APromoter
rs45564433156412150TIM1 g.5383_5397indelins/delExon 4
rs45439103156412024TIM1 g.5509_5511indelins/delExon 4
rs12522248156412004TIM1 g.5529A/GExon 4
rs1553317156412002TIM1 g.5531G/TExon 4
rs1553318156411901TIM1 g.5632C/GIntron 4
rs2270926156402361TIM1 g.15172C/TIntron 5
rs41297581156402282TIM1 g.15251T/CIntron 5
rs41297583156396824TIM1 g.20709C/TIntron 7
rs2036402156396820TIM1 g.20713A/GIntron 7
rs4704837156396790TIM1 g.20743C/AIntron 7
rs41297585156396785TIM1 g.20748T/GIntron 7
rs34670839156392431TIM1 g.25099G/AIntron 8
rs45623443156323275TIMD4 -486indelins/delPromoter
rs41297587156323050TIMD4 -262C/TPromoter
rs6882076156322875TIMD4 -88G/APromoter

Association Test

In this study, the association of the derived rather than minor allele was examined (i.e., the odds ratio was calculated for the derived allele). Eight polymorphisms showed a statistically significant difference in allele frequency between mild and cerebral malaria patients (Table 2 and Figure 1B). Of these, TIM1 -1637G>A, TIM1 -1549G>C and TIM1 -1454G>A, had the smallest P-value (P= 0.0009; odds ratio [OR]= 0.41; 95% confidence interval [CI] 0.24–0.71). The P-value was still significant even after stringent Bonferroni correction (Pc= 0.0009 × 21 × 2 = 0.0378), although the statistical tests are not independent due to LD among polymorphic sites. Since the frequencies of the derived alleles, TIM1 -1637A, TIM1 -1549C, and TIM1 -1454A, were higher in patients with mild malaria than in patients with cerebral malaria, these alleles were regarded as being associated with protection against cerebral malaria. Since these SNPs were in complete LD (r2= 1), in this paper the haplotype comprising TIM1 -1637A, TIM1 -1549C, and TIM1 -1454A is referred to as H2, and the other major haplotype comprising TIM1 -1637G, TIM1 -1549G, and TIM1 -1454G is referred to as H1.

Table 2.  Allele frequencies of polymorphisms in TIM1 and TIMD4
Polymorphism (ancestral allele, followed by derived allele)Mild malaria (2n = 414)Non-cerebral severe malaria (2n = 326)Cerebral malaria (2n = 216)Mild vs non-cerebral severe malaria (P-value, allelic OR, 95% CI)Mild vs cerebral malaria (P-value,allelic OR,95% CI)
rs2116787: 
 T allele364 (88%)288 (88%)197 (91%)NSNS
 C allele50 (12%)38 (12%)19 (9%) 
rs4704844: 
 T allele357 (86%)276 (85%)188 (87%)NSNS
 C allele57 (14%)50 (15%)28 (13%) 
TIM1–1637: 
 G allele336 (81%)277 (85%)197 (91%)NS0.0009
 A allele78 (19%)49 (15%)19 (9%) OR = 0.41, 95% CI 0.24–0.71
TIM1–1569: 
 G allele398 (96%)319 (98%)211 (98%)NSNS
 A allele16 (4%)7 (2%)5 (2%) 
TIM1–1549: 
 G allele336 (81%)277 (85%)197 (91%)NS0.0009
 C allele78 (19%)49 (15%)19 (9%) OR = 0.41, 95% CI 0.24–0.71
TIM1–1454: 
 G allele336 (81%)277 (85%)197 (91%)NS0.0009
 A allele78 (19%)49 (15%)19 (9%) OR = 0.41, 95% CI 0.24–0.71
TIM1 g.5383_5397indel: 
 Insertion90 (22%)88 (27%)66 (31%)NS0.0150
 Deletion324 (78%)238 (73%)150 (69%) OR = 0.63, 95% CI 0.43–0.92
TIM1 g.5509_5511indel:
 Insertion331 (80%)244 (75%)150 (69%)NS0.0032
 Deletion83 (20%)82 (25%)66 (31%) OR = 1.75, 95% CI 1.20—2.56
TIM1 g.5529: 
 A allele354 (86%)281 (86%)203 (94%)NS0.0016
 G allele60 (14%)45 (14%)13 (6%) OR = 0.38, 95% CI 0.20–0.70
TIM1 g.5531: 
 G allele410 (99%)323 (99.08%)216 (100%)NSNS
 T allele4 (1%)3 (0.92%) 
TIM1 g.5632: 
 C allele87 (21%)85 (26%)66 (31%)NS0.0080
 G allele327 (79%)241 (74%)150 (69%) OR = 0.60, 95% CI 0.42–0.88
TIM1 g.15172: 
 C allele369 (89%)305 (94%)206 (95%)0.03590.0084
 T allele45 (11%)21 (6%)10 (5%)OR = 0.56, 95% CI 0.33–0.97OR = 0.39, 95% CI 0.19–0.80
TIM1 g.15251: 
 T allele414 (100%)323 (99.1)216 (100%)NSNS
 C allele 3 (0.9%) 
TIM1 g.20709: 
 C allele400 (97%)320 (98%)210 (97%)NSNS
 T allele14 (3%)6 (2%)6 (3%) 
TIM1 g.20713: 
 A allele348 (84%)281 (86%)191 (88%)NSNS
 G allele66 (16%)45 (14%)25 (12%) 
TIM1 g.20743: 
 C allele410 (97%)318 (98%)214 (99.1%)NSNS
 A allele4 (3%)8 (2%)2 (0.9%) 
TIM1 g.20748: 
 T allele408 (99%)324 (99.4%)212 (98%)NSNS
 G allele6 (1%)2 (0.6%)4 (2%) 
TIM1 g.25099: 
 G allele347 (84%)272 (83%)174 (81%)NSNS
 A allele67 (16%)54 (17%)42 (19%) 
TIMD4 -486indel: 
 Insertion392 (95%)309 (95%)200 (93%)NSNS
 Deletion22 (5%)17 (5%)16 (7%) 
TIMD4 -262: 
 C allele396 (96%)305 (94%)205 (95%)NSNS
 T allele18 (4%)21 (6%)11 (5%) 
TIMD4 -88: 
 G allele310 (75%)235 (72%)152 (70%)NSNS
 A allele104 (25%)91 (28%)64 (30%) 

A SNP, TIM1 g.15172 in intron 5, was found to be associated with protection against severe malaria (P= 0.0359; OR = 0.56; 95% CI 0.33–0.97). However, the P-value was larger than that for polymorphisms that had an association with cerebral malaria. Thus, we focused on the association between polymorphisms and cerebral malaria in the rest of this paper. The results from association tests for the genotype frequency corresponded well to those for allele frequency (Table 2, supplementary Table 3).

The significant association between the promoter polymorphisms of TIM1 and protection against cerebral malaria may have arisen from LD with undetected polymorphisms in the upstream region. To examine this possibility, polymorphisms located between TIM1 and TIM3 were further investigated. Because no polymorphic sites were identified in the region upstream of TIM1 -1637G>A in our variation screening, we analyzed rs2116787 and rs4704844, which were located approximately 8 kb and 13 kb upstream of TIM1 -1637G>A, respectively. Because these SNPs had no statistically significant difference in allele frequency between mild and cerebral malaria patients (Table 2 and Figure 1B), we conclude that the observed association between TIM1 polymorphisms and cerebral malaria is not due to LD with polymorphisms in the TIM3 gene.

LD Structure of TIM1 Gene

Although the TIM1 promoter polymorphisms had the strongest association with cerebral malaria, five polymorphisms, TIM1 g.5383_5397indel, TIM1 g.5509_5511indel, TIM1 g.5529A>G, TIM1 g.5632C>G [c.673 + 49], and TIM1 g.15172C>T [c.782–90], also had a significant association (Table 2 and Figure 1B). We therefore examined, based on the LD structure, whether the observed significant association between these polymorphisms and cerebral malaria was independent of that of the promoter polymorphisms. The pairwise D′ values for 17 polymorphisms around the TIM1 gene region in patients with mild malaria are shown in Figure 1C. All the pairwise D′ values for the above-mentioned polymorphisms and the promoter polymorphisms TIM1 -1637A, TIM1 -1549C, and TIM1 -1454A were at least 0.4 (Figure 1C). Thus, the significant association between the polymorphisms outside the TIM1 promoter region and cerebral malaria may have come from the LD with the TIM1 promoter polymorphisms. Although the present results do not rule out the possibility that these five polymorphisms are associated with cerebral malaria independently of the TIM1 promoter polymorphisms, we conclude that the TIM1 promoter alleles TIM1 -1637A, TIM1 -1549C, and TIM1 -1454A are the most plausible variants responsible for protection against cerebral malaria.

Effect of TIM1 Polymorphisms on Transcription

Two promoter haplotypes (H1 comprises -1637G, -1549G and -1454 G, and H2 comprises -1637A, -1549C and -1454 A) were observed in Thai malaria patients (Table 3), and the H2 haplotype showed significant association with protection against cerebral malaria. To assess the functional significance of the H2 haplotype, allele-specific transcription quantification analysis was conducted. The mean allelic expression ratio of the g.5383_5397Del transcript to the g.5383_5397Ins transcript in H2-Del/H1-Ins subjects was 1.37 (Figure 2), and that in H1-Del/H1-Ins subjects was 0.35, indicating that the level of mRNA transcribed from TIM1 with the H2 promoter haplotype was 3.9 (= 1.37/0.35) times higher than the level of mRNA transcribed from TIM1 with the H1 haplotype (P= 0.004, Mann-Whitney U test). Since the variance for the genomic DNA is small (Figure 2), the present experimental system seems to be reliable.

Table 3.  Estimated haplotype frequencies of TIM1 promoter polymorphisms across three populations
HaplotypePolymorphismEstimated frequency
TIM1 -1637TIM1 -1549TIM1-1454ThaiaHan Chinese (CHB)Yoruba Nigerians (YRI)
  1. aThe frequency was estimated for Thai patients with malaria (n = 478).

H1GGG85%78.9%40.5%
H2ACA15%18.9%3.4%
H3GCA-1.1%44.8%
H4GCG-1.1%-
H5GGA--11.2%
image

Figure 2. Allelic expression ratio of 5383_5397indel transcripts. The allelic expression ratio of g.5383_5397Del to g.5383_5397Ins was obtained by dividing the individual ratio for the RT-PCR products by the mean of the ratio for the PCR products of the genomic DNA. Open circles represent the mean allelic expression ratio for each subject from triplicate experiments. The mean (closed circle) and SD (bar) are presented to the right of the individual values. The P-value was obtained using a Mann-Whitney U-test for comparison of the allelic expression ratio between H2-Del/H1-Ins and H1-Del/H1-Ins subjects. The numbers of subjects are shown in parentheses.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

This study showed that the TIM1 promoter haplotype H2, bearing TIM1 -1637A, TIM1 -1549C and TIM1 -1454A, was strongly associated with protection against cerebral malaria. Allele-specific transcription quantification analysis revealed that mRNA of H2 TIM1 was expressed at a higher level than H1 TIM1. These results suggest that higher expression of TIM1 reduces the risk of developing cerebral malaria. In humans, TIM1 mRNA is expressed at a higher level by Th2 cells than by Th1 cells (Khademi et al. 2004). In mice, in vitro stimulation of CD4+ T cells with a TIM1-specific monoclonal antibody and T cell receptor ligation enhances T cell proliferation, and synthesis of IL-4 is enhanced by this co-stimulation in Th2 cells (Umetsu et al. 2005). Th2 cytokines can inhibit the production of Th1 cytokines, which are thought to be involved in driving the immunopathological process leading to cerebral malaria (Hunt & Grau 2003). Based on these observations, we hypothesize that the higher levels of TIM1 expression associated with the TIM1 promoter haplotype confer protection against cerebral malaria via production of Th2 cytokines, which can inhibit the production of Th1 cytokines. However, further studies are required to determine whether TIM1 molecules are expressed at a low level on Th2 cells in cerebral malaria patients.

The allelic expression ratio in subjects with the H1-Del/H1-Ins diplotype was found to be smaller than 1 (Figure 2), implying that expression of the haplotype with g.5383_5397Del is lower than that of the haplotype with g.5383_5397Ins. Thus, it seems that not only promoter polymorphisms but also polymorphisms in exon 4 affect expression of TIM1. Significant associations between the g.5383_5397indel polymorphism and autoimmune diseases have been reported (Chae et al. 2004; Chae et al. 2003; McIntire et al. 2003). In addition, the possibility that natural selection acts on the variation in exon 4 of TIM1 has been suggested by studying divergence in the TIM1 gene among primates and the polymorphisms in human populations (Nakajima et al. 2005). These phenomena may have occurred because of differences in the expression levels of TIM1 between haplotypes with different variants in exon 4.

Potential regulatory elements in the promoter region of the TIM1 gene were identified by using TFSEARCH program version 1.3, which identifies highly correlated sequence fragments against the TFMATRIX transcription factor binding site profile database in the ‘TRANSFAC’ databases (Heinemeyer et al. 1998). To avoid false negative results, the threshold was set at a relatively low score of 75.0. We found that TIM1 -1454G>A was located in the potential transcription factor binding site for CCAAT box binding protein-1 (CTF-1, also called NF-1). CTF-1 is a proline-rich transcription factor that selectively activates transcription of genes containing the TGGCNNNNNGCCAAT consensus sequence in the promoter region. The CTF-1 consensus sequence is more similar to the sequence of the TIM1 -1454A derived allele (AGCCNNNNNGCCCAG) than that of the TIM1 -1454G ancestral allele (AGCCNNNNNGCCCGG). Although the transcription factor that binds to the TIM1 promoter remains to be studied, the higher expression level of TIM1 with the H2 promoter haplotype comprising TIM1 -1454A, as observed in the allele-specific transcription quantification analysis, corresponds well with the presence of a putative CTF-1 transcription factor binding site. The derived allele, TIM1 -1454A, may create a CTF-1 binding site and enhance TIM1 expression.

Because of the complete LD between the TIM1 -1637G>A, TIM1 -1549G>C and TIM1 -1454G>A polymorphisms in Thai population, the primary variant responsible for protection against cerebral malaria could not be statistically determined in this study. The haplotype frequencies of TIM1 promoter polymorphisms in the Yoruba Nigerian population were different from those of the Thai and Han Chinese population (Table 3). Thus, association studies in African populations require to be conducted to detect a primary variant showing the smallest P-value among three polymorphisms.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

We sincerely thank the patients with malaria and the healthy volunteers who participated in this study. We also thank Dr Kazuya Omi for valuable comments and suggestions; Ms Miwa Takasu for assistance with carrying out RT-PCR; Dr Nao Nishida for assistance with analysis using the Agilent 2100 BioAnalyzer; and Associate Professor Duangporn Nakapunchai for kind assistance with collecting blood samples from the healthy subjects. We thank two anonymous reviewers for valuable comments and suggestions on this manuscript. This study was supported by the Thailand Research Fund through the Royal Golden Jubilee PhD programme (Grant No.PHD/0047/2544) to J.P. and P.N. The study was also supported in part by the Core University System Exchange Programme of the Japan Society for the Promotion of Science (coordinated by the University of Tokyo and Mahidol University), the National Research Council of Thailand, a Mahidol University grant, and KAKENHI Grant-in-Aid for Scientific Research on Priority Areas “Comprehensive Genomics” and “Applied Genomics” from the ministry of Education, Culture, Sports, Science and Technology of Japan.

Conflict of Interest Statement.  None declared

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • Barrett, J. C., Fry, B., Maller, J. & Daly, M. J. (2005) Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 21, 263265.
  • Chae, S.-C., Song, J.-H., Shim, S.-C., Yoon, K.-S. & Chung, H.-T. (2004) The exon 4 variations of Tim-1 gene are associated with rheumatoid arthritis in a Korean population. Biochemical and Biophysical Research Communications 315, 971975.
  • Chae, S., Song, J., Lee, Y., Kim, J. & Chung, H. (2003) The association of the exon 4 variations of TIM1 gene with allergic disease in Korean population. Biochemical and Biophysical Research Comunication 312, 346350.
  • Feigelstock, D., Thompson, P., Mattoo, P. & Kaplan, G. G. (1998a) Polymorphisms of the hepatitis A virus cellular receptor 1 in African green monkey kidney cells result in antigenic variants that do not react with protective monoclonal antibody 190/4. J Virol 72, 62186222.
  • Feigelstock, D., Thompson, P., Mattoo, P., Zhang, Y. & Kaplan, G. G. (1998b) The human homolog of HAVcr-1 codes for a hepatitis A virus cellular receptor. J Virol 72, 66216628.
  • Han, W. K., Bailly, V., Abichandani, R., Thadhani, R. & Bonventre, J. V. (2002) Kidney Injury Molecule-1 (KIM-1): a novel biomarker for human renal proximal tubule injury. Kidney Int 62, 237244.
  • Heinemeyer, T., Wingender, E., Reuter, I., Hermjakob, H., Kel, A. E., Kel, O. V., Ignatieva, E. V., Ananko, E. A., Podkolodnaya, O. A., Kolpakov, F. A., Podkolodny, N. L. & Kolchanov, N. A. (1998) Databases on transcriptional regulation: TRANSFAC, TRRD and COMPEL. Nucleic Acids Res 26, 362367.
  • Hill, A. V. (1999) The immunogenetics of resistance to malaria. Proc Assoc Am Physicians 111, 272277.
  • Hunt, N. H. & Grau, G. E. (2003) Cytokines: accelerators and brakes in the pathogenesis of cerebral malaria. Trends in Immunology 24, 491499.
  • Ichimura, T., Bonventre, J. V., Bailly, V., Wei, H., Hession, C. A., Cate, R. L. & Sanicola, M. (1998) Kidney injury molecule-1 (KIM-1), a putative epithelial cell adhesion molecule containing a novel immunoglobulin domain, is up-regulated in renal cells after injury. J Biol Chem 273, 41354142.
  • Kaplan, G., Totsuka, A., Thompson, P., Akatsuka, T., Moritsugu, Y. & Feinstone, S. M. (1996) Identification of a surface glycoprotein on African green monkey kidney cells as a receptor for hepatitis A virus. Embo J 15, 42824296.
  • Khademi, M., Illes, Z., Gielen, A. W., Marta, M., Takazawa, N., Baecher-Allan, C., Brundin, L., Hannerz, J., Martin, C., Harris, R. A., Hafler, D. A., Kuchroo, V. K., Olsson, T., Piehl, F. & Wallstrom, E. (2004) T Cell Ig- and mucin-domain-containing molecule-3 (TIM-3) and TIM-1 molecules are differentially expressed on human Th1 and Th2 cells and in cerebrospinal fluid-derived mononuclear cells in multiple sclerosis. J Immunol 172, 71697176.
  • Kimura, R., Nishioka, T., Soemantri, A. & Ishida, T. (2004) Cis-acting effect of the IL1B C-31T polymorphism on IL-1 beta mRNA expression. Genes Immun 5, 572575.
  • Kimura, R., Nishioka, T., Soemantri, A. & Ishida, T. (2005) Allele-specific transcript quantification detects haplotypic variation in the levels of the SDF-1 transcripts. Hum Mol Genet 14, 15791585.
  • McIntire, J. J., Umetsu, S. E., Macaubas, C., Hoyte, E. G., Cinnioglu, C., Cavalli-Sforza, L. L., Barsh, G. S., Hallmayer, J. F., Underhill, P. A., Risch, N. J., Freeman, G. J., DeKruyff, R. H. & Umetsu, D. T. (2003) Immunology: hepatitis A virus link to atopic disease. Nature 425, 576.
  • Meyers, J. H., Chakravarti, S., Schlesinger, D., Illes, Z., Waldner, H., Umetsu, S. E., Kenny, J., Zheng, X. X., Umetsu, D. T., DeKruyff, R. H., Strom, T. B. & Kuchroo, V. K. (2005) TIM-4 is the ligand for TIM-1, and the TIM-1-TIM-4 interaction regulates T cell proliferation. Nat Immunol 6, 455464.
  • Monney, L., Sabatos, C. A., Gaglia, J. L., Ryu, A., Waldner, H., Chernova, T., Manning, S., Greenfield, E. A., Coyle, A. J., Sobel, R. A., Freeman, G. J. & Kuchroo, V. K. (2002) Th1-specific cell surface protein Tim-3 regulates macrophage activation and severity of an autoimmune disease. Nature 415, 536541.
  • Nakajima, T., Wooding, S., Satta, Y., Jinnai, N., Goto, S., Hayasaka, I., Saitou, N., Guan-Jun, J., Tokunaga, K., Jorde, L. B., Emi, M. & Inoue, I. (2005) Evidence for natural selection in the HAVCR1 gene: high degree of amino-acid variability in the mucin domain of human HAVCR1 protein. Genes Immun 6, 398406.
  • Sabatos, C. A., Chakravarti, S., Cha, E., Schubart, A., Sanchez-Fueyo, A., Zheng, X. X., Coyle, A. J., Strom, T. B., Freeman, G. J. & Kuchroo, V. K. (2003) Interaction of Tim-3 and Tim-3 ligand regulates T helper type 1 responses and induction of peripheral tolerance. Nat Immunol 4, 11021110.
  • Sanchez-Fueyo, A., Tian, J., Picarella, D., Domenig, C., Zheng, X. X., Sabatos, C. A., Manlongat, N., Bender, O., Kamradt, T., Kuchroo, V. K., Gutierrez-Ramos, J. C., Coyle, A. J. & Strom, T. B. (2003) Tim-3 inhibits T helper type 1-mediated auto- and alloimmune responses and promotes immunological tolerance. Nat Immunol 4, 10931101.
  • The International HapMap Consortium. (2003) The International HapMap Project. Nature 426, 789796.
  • Umetsu, S. E., Lee, W.L., McIntire, J. J., Downey, L., Sanjanwala, B., Akbari, O., Berry, G. J., Nagumo, H., Freeman, G. J., Umetsu, D. T. & DeKruyff, R. H. (2005) TIM-1 induces. T cell activation and inhibits the development of peripheral tolerance. Nat Immunol 6, 447454

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Table S1. Method used to genotype the TIM 1 and TIM D4 genes

Table S2. Primers used for allele-specific transcript quantification

Table S3. Genotype frequencies of polymorphisms in TIM 1 and TIM D4.

Please note: Blackwell Publishing are not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

FilenameFormatSizeDescription
AHG_424_sm_TablesS1-S3.doc45KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.