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Association of CTLA4 gene polymorphisms with lymphatic filariasis in an East Malaysian population
Human Immunology 72 (2011) 607-612
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Association of CTLA4 gene polymorphisms with lymphatic filariasis in an East Malaysian population Zulkarnain Md Idris a, Noorizan Miswan a, Jamail Muhi b, Tajul Ariffin Awang Mohd c, Ju¨rgen F.J. Kun d, Rahmah Noordin a,* a
Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang, Malaysia Sarawak Health Department, Kuching, Sarawak, Malaysia c Sandakan Health Office, Sandakan, Sabah, Malaysia d Institute for Tropical Medicine, University of Tu ¨ bingen, Tu ¨ bingen, Germany b
A R T I C L E
I N F O
Article history: Received 2 November 2010 Accepted 31 March 2011 Available online 8 April 2011
Keywords: CTLA4 gene Lymphatic filariasis Polymorphisms
A B S T R A C T
Lymphatic filariasis (LF) is a parasitic disease caused by threadlike worms of the Brugia and Wuchereria species that live in the human lymphatic system. Regulatory T cells (Tregs) may play a key role in the pathogenesis of LF, and cytotoxic T-lymphocyte antigen-4 (CTLA4) expressed by Tregs is a potential candidate gene because it modulates T-cell activation. A case– control study was performed to establish a potential association of 5 CTLA4 gene promoter single nucleotide polymorphisms (SNPs; rs733618, rs11571316, rs5742909, rs231775, and rs16840252) with the occurrence of LF in an East Malaysian population (320 LF-infected individuals and 150 healthy controls). Polymorphisms were evaluated using TaqMan real-time polymerase chain reaction followed by direct sequencing. LF carriers of the rs733618 AG genotypes (p ⫽ 0.02) and those with combined minor allele G carriers (AG ⫹ GG; p ⫽ 0.01) exhibited a significantly decreased risk for LF. Among the asymptomatic amicrofilaremic cases, positive associations were reported for all genotypes and variants of rs733618 with odds ratios (ORs) ranging from 0.27 to 0.45. In the asymptomatic microfilaremic cases, marker rs231775 exhibited a significant decreased risk, with ORs ranging from 0.50 to 0.57. The study has identified SNPs in the CTLA4 promoter gene that may be functionally linked with susceptibility to LF. 䉷 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction Lymphatic filariasis (LF) is caused by the vector-borne helminth parasites Wuchereria bancrofti, Brugia malayi, and Brugia timori and is transmitted by mosquitoes [1]. Ninety percent of LF is caused by W bancrofti, which occurs throughout the tropics and subtropics. B malayi infects about 13 million people in south and southeast Asia, whereas B timori is restricted to Timor Leste and a few islands in Indonesia [2]. Adult worms reside in the lymphatics and lymph nodes and induce changes that result in dilatation of lymphatics and thickening of the lymphatic vessel walls. Progressive lymphatic damage and pathology result from the summation of the effect of tissue alterations induced by both living and nonliving adult parasites, the host inflammatory response to the parasites and their secreted antigens, and those seen because of secondary bacterial or fungal infections [3]. A total of 44 million people suffer from 1 or more of the overt manifestations of the infection: lymphedema and elephantiasis of the limbs or genitals, hydrocele, chyluria, pneumonitis, or recurrent infections associated with damaged lymphatics.
* Corresponding author. E-mail address: [email protected] (R. Noordin).
In addition, a significant percentage of infected individuals are outwardly asymptomatic and have no circulating microfilaria but have serologic and radiological evidence of the so-called “cryptic infection” [4,5]. Filarial infections characteristically are associated with a profound downregulated T-cell response to parasite antigen in infected individuals compared with filaria-exposed but uninfected individuals [6]. In essence, some cells of the immune system act in a suppressive or downregulatory manner to switch off inflammatory and protective immune responses mediated by macrophages and eosinophils. These cells, termed regulatory T cells (Tregs), were first defined as inhibiting autoimmune reactions, and such cells appeared to develop naturally in both humans and mice [7]. The regulatory cells release soluble suppressive cytokines (interleukin [IL]-10 and transforming growth factor ), which bind receptors on effector lymphocytes with the effect of deactivating immune cells. The regulatory cells also express a surface molecule, cytotoxic Tlymphocyte antigen-4 (CTLA4) protein, coded by the CTLA4 (CD152) gene, which is stored in endosomal compartments and expressed on the cell surface following activation [8] and delivers an inhibitory signal to T cells in contrast to its homologue CD28. In humans, filarial infections (LF and onchocerciasis) have long been associated
0198-8859/11/$32.00 - see front matter 䉷 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2011.03.017
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with IL-10 and transforming growth factor  and regulatory phenotype T-cell clones have been isolated from onchocerciasis patients [9]. In human LF, infected patients exhibit higher levels of CTLA4 expression in peripheral blood T cells than do uninfected people, and in vitro IL-5 responses (associated with protection) are significantly enhanced in the presence of anti-CTLA4 antibody [10]. Human CTLA4 is encoded by a gene in chromosome 2p33 in a region also containing inducible T-cell costimulator and CD28 genes and shares with the latter a high nucleotide identity, strongly suggesting that they are the result of a gene duplication [11]. Many single nucleotide polymorphisms (SNPs) have been identified in the CTLA4 region [12,13]. SNPs in the promoter region can affect the binding affinity of transcriptional factor involved in the regulation of gene expression [14]. We hypothesized that potentially functional CTLA4 promoter SNPs and haplotypes could alter transcriptional activity, thus affecting susceptibility to develop LF, and might explain some of the between-person differences in LF manifestations. To date, no study has comprehensively investigated the CTLA4 promoter SNPs on their functional roles and contributions to the risk of developing LF. To test our hypothesis, polymorphisms located in the CTLA4 promoter were identified from the public dbSNP (http:// www.ncbi.nlm.nih.gov/projects/SNP/) database. Four promoter polymorphisms, rs733618 (⫺1,722 A/G), rs11571316 (⫺1,577 C/T), rs5742909 (⫺319 C/T), and rs16840252 (⫺1,477 C/T), and exon 1 SNP (i.e., rs231775 [⫹48 A/G]) were studied by allelic discrimination genotyping assay. 2. Subjects and methods The study protocol complied with the Declaration of Helsinki and was approved by the Committee on the Ethics of Human Research of Universiti Sains Malaysia. Written informed consent was obtained from each participant. 2.1. Subjects The study population comprised a total of 320 individuals (179 males and 141 females) from LF-endemic areas. The ages ranged from 3 to 78 years (28.23 ⫾ 19.68 years) and subjects were recruited from various districts from the state of Sarawak between September 2004 until March 2006 and the state of Sabah in December 2008. Nocturnal subperiodic B malayi is the species prevalent in these areas. Three rounds of mass drug administration (MDA) have been performed in this area with pre-MDA microfilaria (mf) prevalence of 2% (20/1,000 population). The mf prevalence at the time of the study was 2.64% (32/1,211) from 16 villages; this unchanged mf prevalence rate was probably caused by treatment noncompliance or insufficient MDA coverage because of the logistic challenges in this area. A resident of the endemic area was defined as someone who has lived in the community for at least 10 years and who has not been absent for more than 6 months. Children younger than 10 years were confirmed as residents who have lived in the community since birth. The age prevalence curve increased from under 3% in the age group 3 to 5 years to near 30% between ages 11 and 20 and then declined after the age of 20. The prevalence among middle-age adults was less than 20%, whereas the prevalence between genders was not significantly different.
All individuals are native people of East Malaysia, with the majority of Malay ethnicity, and some are indigenous people of Sabah (Kadazan and Bajau) and Sarawak (Iban and Melanau). However, these ethnic differences are not considered significant. For all patients with LF, the diagnostic test for LF infection was performed using the traditional thick blood smear and pooled real-time polymerase chain reaction (PCR; pooled blood spot samples from 10 individuals per pool) for the presence of mf and BRUGIArapid. The rapid test uses BmR1 recombinant antigen that detects specific antifilarial immunoglobulin (Ig)-G4 antibodies in individuals infected with B malayi and B timori [15–17]. Elevated levels of antifilarial IgG4 antibodies are a marker of active LF [18 –20]. The screening was based on household survey. In the morning, houseto-house visits were made. The villagers were given a personal tag on their wrists and were asked about their personal information (demographic data), drug (diethylcarbamazine ⫹ albendazole) intake, bednet use, and symptoms. The location of each household was recorded by a global positioning system. Villagers were then asked to gather at a community hall or village head’s house at 8 PM for sample collection. A custom-made barcoding system was used to label all wrist tags, blood tubes, slides, rapid test cassettes, and aliquots. The individuals with LF were divided into 2 groups, namely, asymptomatic microfilaremia (AM; i.e., AM/mf⫹/IgG4⫹) and asymptomatic amicrofilaraemia (AsAm; i.e., AsAm/mf⫺/ IgG4⫹). Asymptomatic individuals were assessed based on an examination of their limbs to look for symptoms of edema (limb enlargement) and direct questions regarding LF symptoms (edema, lymphangitis, fever) by field staff who spoke in the local dialect. The AM group consisted of 109 individuals (26.01 ⫾ 20.02 years) and was characterized by the presence of mf in 60 l of finger-prick blood and positive result by the rapid test. The AsAm group comprised 211 individuals (29.28 ⫾ 19.48 years) who were negative by thick blood smear, positive by the rapid test, and negative by the pooled real-time PCR. The healthy endemic control group of uninfected people consisted of 150 individuals (64 males and 86 females) aged 3 to 70 years (21.3 ⫾ 16.29 years) who were negative by all diagnostic methods and were selected from community volunteers from the same district. 2.2. SNP selection and genotyping The identification of SNPs associated with LF was performed by examining the relationship of promoter SNPs of the CTLA4 gene to LF. Using public databases, including PubMed (National Center for Biotechnology Information) and Online Mendelian Inheritance in Man, 5 SNPs were selected. Based on the previous reports, these SNPs may be expected to result in a change of function or expression of the encoded protein [13,21]. Genomic DNA was extracted from blood spots of each participant using a QIAamp mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. All SNPs were genotyped using a TaqMan assay in a 72-well rotor format (Corbett Research, New South Wales, Australia). All probes and primers were designed by the Assay-by-Demand service from Applied Biosystems (Foster City, CA; Table 1). Genotyping was performed in 20-l reactions comprising 10 ng of genomic DNA, 1⫻ TaqMan SNP genotyping assay (consisting of 200 nM TaqMan MGB probes, FAM and VIC
Table 1 Positions, nucleotide variations, and assay ID for CTLA4 DbSNP ID
Chromosome position
Relative position/region
Allele
Assay ID (Applied Biosystems)
rs733618 rs11571316 rs16840252 rs5742909 rs231775
Chr2q33: 204439189 Chr2q33: 204439334 Chr2q33: 204439764 Chr2q33: 204440592 Chr2q33: 204440959
⫺1,722/promoter ⫺1,577/promoter ⫺1,147/promoter ⫺319/promoter ⫹48/exon 1 (17 T/A)
A/G C/T C/T C/T A/G
C_2415791_10 C_30981395_10 C_32900355_10 C_27834180_10 C_2415786_20
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dye-labeled, and 900 nM of primers), and 2⫻ HotStarTaq Master Mix (containing 1.5 mM MgCl2, 200 M dNTPs, HotStarTaq DNA polymerase, and RNase-free water; Qiagen, Hilden, Germany). Real-time PCR reactions were thermally cycled with an initial denaturation step at 95⬚C for 15 minutes, followed by 40 cycles of 94⬚C for 30 seconds, 60⬚C for 1 minute, and 72⬚C for 1 minute and then a final extension step at 72⬚C for 10 minutes. Real-time fluorescence detection was performed during the annealing/extension step of each cycle. To ensure genotyping quality, positive control and nontemplate negative controls were included for each genotype in each run. Allelic discrimination was performed on a Rotor-Gene 6000 Multiplex System and data analysis was conducted with the software interface version 1.7. Subsequently, randomly selected samples were genotyped through direct sequencing for genotype confirmation and 100% concordance was obtained for every condition. Nucleotide changes were detected by visual inspection of chromatograms. 2.3. Statistical analysis Statistical analysis was performed using PASW Statistic software 17.0 (SPSS, Inc., Chicago, IL). Allele and genotype frequencies were calculated on LF and control subjects by direct counting. The CTLA4 SNPs were tested for Hardy–Weinberg equilibrium (HWE) by comparing observed and expected genotype frequencies in both LF and control groups using the 2 test (p value ⬎ 0.05 was considered consistent with HWE). The associations of polymorphisms were estimated by logistic regressions analysis with odds ratios (OR) and 95% confidence intervals (95% CI). The level of statistical significance was set at a p value less than 0.05 (2-tailed test). For multiple marker association analyses, the estimation of pairwise linkage disequilibrium (LD) values and haplotype block structure was performed using the default method of Gabriel et al. [22] and displayed schematically with their population frequencies and interblock connections based on the Haploview program output. Haplotype analysis was performed using SNPAnalyzer version 2.0 (http:// snp.istech21.com/snpanalyzer/2.0/).
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3. Results Five SNPs (i.e., rs733618, rs11571316, rs16840252, rs5742909, and rs231775) in the CTLA4 gene promoter and exon 1 region were genotyped in 320 individuals (109 AM and 211 AsAM) with LF, as well as in 150 endemic control subjects. The frequency of the genotypes in the LF group followed HWE. Two markers, namely, rs733618 and rs11571316, demonstrated borderline significant deviation (p ⫽ 0.01– 0.05) from HWE (underlined in Table 2), but no marker had an HWE p value less than 0.05 in more than 1 dataset, so all markers were used for the association analysis. Individual SNPbased association analysis revealed a number of significant associations between several SNPs and disease manifestations in the LF samples (Table 2). Logistic regression analysis demonstrated that the LF carriers of the rs733618 AG genotypes and those with combined minor allele G carriers (AG ⫹ GG) exhibited a significant decrease risk for LF (OR ⫽ 0.61, p ⫽ 0.02, 95% CI ⫽ 0.41– 0.91; and OR ⫽ 0.61, p ⫽ 0.01, 95% CI ⫽ 0.41– 0.90, respectively). No significant relationships between any other SNPs were observed. However, among the AsAm group, positive associations with disease were observed for all genotypes and variants of rs733618, with ORs ranging from 0.27 (95% CI ⫽ 0.09 – 0.84, p ⫽ 0.02, for the GG genotype) to 0.45 (95% CI ⫽ 0.29 – 0.70, p ⫽ 0.001, for the AG genotype). In the AM group, marker rs231775 exhibited a significant decrease in risk and directly correlated with disease manifestation, with ORs ranging from 0.50 (95% CI ⫽ 0.15– 0.75, p ⫽ 0.008, for the GG genotype) to 0.57 (95% CI ⫽ 0.33– 0.97, p ⫽ 0.04, for the AG genotype). No significant associations were observed for the other SNPs in the AM cases. Four SNPs, namely, rs733618, rs11571316, rs16840252, and rs5742909, within the LF haplotype block gave 9 different haplotype combinations (Table 3). Four haplotypes gave more than 5% in frequencies, ATCC, GCCC, ACCC, and ACTT, and at least 3 major alleles were found in each genotype. The ATCC haplotype demonstrated the highest frequencies, with 39.7%. In LF disease manifestations, both SNPs rs11571316 and rs16840252 were defined in the
Table 2 Association of CTLA4 polymorphisms with LF and disease manifestation in the East Malaysian population SNP
rs733618
HWE rs11571316
HWE rs16841252
HWE rs5742909
HWE rs231775
HWE
Genotype
AA AG GG AG ⫹ GG CC CT TT CT ⫹ TT CC CT TT CT ⫹ TT CC CT TT CT ⫹ TT AA AG GG AG ⫹ GG
Control n (%)
66 (44.0) 75 (50.0) 9 (6.0) 84 (56.0) 0.04 48 (32.0) 85 (56.7) 17 (11.3) 102 (68.0) 0.02 121 (80.7) 29 (19.3) 0 (0.0) 29 (19.3) 0.19 121 (80.7) 29 (19.30 0 (0.0) 29 (19.3) 0.19 52 (34.6) 70 (46.7) 28 (18.7) 98 (65.3) 0.61
LF (AM and AsAsm)
AM
n (%)
OR
95% CI
p value
180 (56.3) 125 (39.0) 15 (4.7) 140 (43.8)
1.00 (RV) 0.61 0.61 0.61
0.41–0.91 0.26–1.47 0.41–0.90
0.02 0.27 0.01 0.25
106 (33.1) 167 (52.2) 47 (14.7) 214 (66.9)
1.00 (RV) 0.89 1.25 0.95
0.58–1.37 0.65–2.40 0.63–1.44
0.59 0.50 0.81 0.15
247 (77.2) 69 (21.6) 4 (1.2) 73 (22.8)
1.00 (RV) 1.17 — 1.23
0.72–1.89 — 0.76–2.00
0.54 — 0.39 0.65
251 (78.4) 64 (20.0) 5 (1.6) 69 (21.7)
1.00 (RV) 1.06 — 1.14
0.65–1.74 — 0.71–1.86
0.80 — 0.58 0.69
128 (40.0) 145 (45.3) 47 (14.7) 192 (60.0)
1.00 (RV) 0.84 0.68 0.80
0.55–1.29 0.39–1.20 0.53–1.19
0.43 0.19 0.27 0.57
AsAm
n (%)
OR
95% CI
p value
44 (40.4) 55 (50.5) 10 (9.1) 65 (59.6)
1.00 (RV) 1.10 1.67 1.16
0.67–1.84 0.63–4.43 0.70–1.91
0.72 0.31 0.56 0.22
43 (39.4) 57 (52.3) 9 (8.3) 66 (60.6)
1.00 (RV) 0.75 0.59 0.72
0.44–1.27 0.24–1.46 0.43–1.21
0.29 0.26 0.22 0.10
93 (85.3) 15 (13.9) 1 (1.0) 16 (14.7)
1.00 (RV) 0.63 — 0.72
0.34–1.33 — 0.37–1.34
0.25 — 0.33 0.10
93 (85.3) 14 (12.8) 2 (1.8) 16 (14.7)
1.00 (RV) 0.63 — 0.72
0.32–1.26 — 0.37–1.40
0.19 — 0.33 0.11
56 (51.4) 43 (39.4) 10 (9.2) 53 (48.6)
1.00 (RV) 0.57 0.33 0.50
0.33–0.97 0.15–0.75 0.30–0.83
0.04 0.008 0.007 0.68
n (%)
OR
95% CI
p value
136 (64.5) 70 (33.2) 5 (2.3) 75 (35.5)
1.00 (RV) 0.45 0.27 0.43
0.29–0.70 0.09–0.84 0.28–0.67
0.001 0.02 0.001 0.25
63 (29.9) 110 (52.1) 38 (18.0) 148 (70.1)
1.00 (RV) 0.99 1.70 1.11
0.62–1.58 0.86–3.38 0.70–1.74
0.95 0.13 0.66 0.40
154 (73.0) 54 (25.6) 3 (1.4) 57 (27.0)
1.00 (RV) 1.46 — 1.54
0.88–2.43 — 0.93–2.56
0.14 — 0.09 0.48
158 (74.9) 50 (23.7) 3 (1.4) 53 (25.1)
1.00 (RV) 1.32 — 1.40
0.79–2.21 — 0.94–2.33
0.29 — 0.20 0.67
72 (34.1) 102 (48.3) 37 (17.5) 139 (65.9)
1.00 (RV) 1.05 0.95 1.02
0.66–1.68 0.52–1.75 0.66–1.60
0.83 0.88 0.92 0.93
AM ⫽ asymptomatic microfilaremic; AsAm ⫽ asymptomatic amicrofilaremic; 95% CI ⫽ 95% confidence interval; OR ⫽ odds ratio; RV ⫽ reference value; HWE ⫽ Hardy–Weinberg equilibrium.
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Table 3 Haplotype frequencies in an LF population Haplotype frequencies Block 1 LF SNP Allele Hap 1 Hap 2 Hap 3 Hap 4 Hap 5 Hap 6 Hap 7 Hap 8 Hap 9 Block 1 AM SNP Allele Hap 1 Hap 2 Hap 3 Block 1 AsAm SNP Allele Hap 1 Hap 2 Hap 3 Hap 4 Hap 5
rs733618 A/G A G A A A G A G G
rs11571316 C/T T C C C C T C T C
rs11571316 C/T C T C
rs16840252 C/T C C T
rs11571316 C/T T C C C C
rs16840252 C/T C C T T C
rs16840252 C/T C C C T T C C C T
rs5742909 C/T C C C T C T T C C
0.397 0.255 0.223 0.104 0.007 0.003 0.003 0.003 0.001
0.547 0.344 0.078 rs5742909 C/T C C T C T
0.441 0.412 0.130 0.012 0.005
AM ⫽ asymptomatic microfilaremic; AsAm ⫽ asymptomatic amicrofilaremic; LF ⫽ lymphatic filariasis.
same haplotype blocks for AM and AsAm, whereas an additional SNP, rs5742909, was reported only in the AsAm haplotype block. All haplotypes (3 AM haplotypes and 5 AsAm haplotypes) were at least or more than 5%, and the CC and TCC haplotype gave the highest frequency distribution, with 54.7 and 44.1% in AM and AsAm, respectively. 4. Discussion Commonly known as elephantiasis, LF is a devastating parasitic infection transmitted by mosquitoes. Less than half of the infected people are clinically symptomatic, yet all have hidden damage to their lymphatic and/or renal systems. This state of asymptomatic microfilaremia is associated with a highly downregulated immune system [23], but it is unclear how, when, or even whether these individuals will progress to develop 1 or more overt clinical manifestations of filarial disease. With the circulating mf in their blood, they are an important source of infection. A second asymptomatic presentation is termed AsAm. In the endemic areas, a proportion of the population does not exhibit mf or clinical manifestation although they are exposed to infected larva at a rate similar to that of individuals who became microfilaremic. Their infections are defined not by microfilaremia, but by the presence of parasite antigen or antifilarial IgG4 antibodies in the blood. CTLA4 plays an essential role in immunologic homeostasis as a negative regulator of T-cell activation. It is highly expressed by Tregs and could play an important role in their function [24]. A number of studies have demonstrated that polymorphisms, especially in the CTLA4 gene promoter region, are associated with autoimmune, inflammatory, and noninfectious diseases such as ankylosing spondylitis [25–29]. However, such an association, especially in promoter polymorphisms of the CTLA4 gene in helminth human infections, has not been well investigated. Our study demonstrated that rs231775 (⫹49) minor allele G carriers and genotype among AM individuals exhibited a positive association with LF in the East Malaysian population. Previous studies revealed that the CTLA4 rs231775 (⫹49) variation has a
functional effect on the expression level of CTLA4 [30,31]. The rs231775 (⫹49) G allele has been associated with increased risk for diseases resulting from an overly vigorous immune response, including insulin-dependent diabetes mellitus and autoimmune disorders. Likewise, in an earlier study this allele was also associated with improved clearance of hepatitis C virus infection resulting from interferon ␣– based therapy [32]. These studies could not rule out the role of other SNPs linked to ⫹49 because the other haplotype-tagging SNPs were not studied. The A-to-G mutation at position ⫹49 leads to a nonsynonymous amino acid change from threonine to alanine, thus changing the polarity of the amino acid and potentially altering the function of the protein in the signal peptide. Lymphocytes from donors carrying the rs231775 (⫹49) G allele appeared to express less CTLA4 on their surfaces, proliferate more under conditions of suboptimal activation, and exhibit less CTLA4-mediated inhibition of T-cell responses [30,31]. A study by Anjos et al. indicated that the predisposing Ala allele (⫹49G) is incompletely glycosylated in the endoplasmic reticulum, leading to retrograde transport of a portion of the molecules to cytoplasm for degradation [33]. They also demonstrated that persons with homozygous GG have one-third less CTLA4 on their T-cell surface than people with homozygous AA. This ultimately results in less mature CTLA4 (Ala) at the cell surface, which may explain in part the reduced inhibitory function of CTLA4 reported in individuals with the ⫹49G allele [31]. With regard to infectious disease, a study including 391 individuals in a cohort from Finland demonstrated a significant difference between Helicobacter pylori seropositive and negative individuals in enzyme catalyzed oxidative degradation of tryptophan activity only in rs231775 (⫹49) G allele carriers [34]. This finding also agrees with that of FernÂndez-Mestre et al., who demonstrated that the rs231775 (⫹49) allele and genotype were associated with a risk of parasitic disease [21]. The analysis involved different clinical groups of patients infected with Leishmania and demonstrated an increased frequency of heterozygous AG genotype in patients with diffused cutaneous leishmaniasis compared with patients with localized cutaneous leishmaniasis and intermediate cutaneous leishmaniasis. This indicated that the heterozygous genotype associated with overactivation of T-cell proliferation could confer susceptibility to the development of the more severe clinical form of cutaneous leishmaniasis [21]. In addition, the homozygous GG genotype of rs231775 (⫹49) has been reported to be associated with hepatitis C virus infection [32]. Similarly, the logistic regression model by Hu et al. demonstrated that the CTLA4 rs231775 (⫹49) variant genotype GG was associated with an increased risk for 2 virus infection–related cancers, namely, hepatocellular carcinoma and cervical cancer, by 1.46-fold and 1.55-fold when compared with common homozygous AA [35]. The present study demonstrated that rs733618 (⫺1,722) minor allele carriers and genotypes had a positive association with LF (AM and AsAm), as well as with AsAm, but not with AM. Thus, this may imply that individuals with rs733618 (⫺1,722) SNP may have a predisposition to become AsAm. Thio et al. also suggest a role for the promoter SNP rs 733618 (⫺1,722) in hepatitis B virus (HBV) recovery [36]. Its strong association may be the result of its linkage with the SNP rs231775 (⫹48), because rs733618 (⫺1,722) haplotype combinations were only associated with the G allele of rs231775 (⫹49). Alternatively, because it is located in the promoter region of the gene, it may alter the transcriptional regulation of CTLA4. If rs733618 (⫺1,722) has an independent association, the data would support decreased CTLA4 production with the rs733618C allele, leading to an increased chance of recovery from HBV infection [36]. Previous studies have demonstrated that the functional effect of genetic variation may lead to interindividual differences in immune response capacity [37]. These studies were supported by the
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association of several known SNPs in immunomodulatory genes and altered immune capacity [38]. Thus, given that parasite infection may in part depend on escaping immune surveillance, the SNPs rs733618 (⫺1,722) and rs231775 (⫹49) may constitute risk factors for susceptibility to parasitic infections. In the present study, the results obtained by specific antibody and immunomodulatory genes such as CTLA4 (expressed by Treg) may suggest that the interindividual differences contributed to disease susceptibility. In this study, both AM and AsAm types of LF asymptomatic individuals have elevated IgG4 antibodies compared with healthy controls. It has been suggested that the activation of Treg cells in asymptomatic carriers keeps the pathology mild enough to avoid symptoms [39]. Unlike other IgG subclasses, IgG4 is found under conditions where IL-10 produced by Tregs is increased after chronic helminth infections [40]. Furthermore, a recent study reported that an intermediate degree of correlation indicated the induction of IgG4 antibody also involved cell– cell contact with CTLA4 in CD4⫹CD2⫹ Tregs [9]. In addition, CTLA4 rs231775 (⫹49) has demonstrated an association with autoimmune pancreatitis, which is characterized serologically by an increased IgG4 level [41]. One haplotype block was identified for each cohort from the LD structure. Associated SNP rs733618 was found to be located in the block structure of the LF cohort but none of the associated SNPs (rs733618 and rs231775) was found in the block structure when LF types were computed. Interestingly, consistently strong LD (D= and r2) was reported between rs16840252 and rs5742909 across all LD structures (data not shown). In the presence of 2 functional polymorphisms on the same LD block, it is possible that the predisposing allele of 1 polymorphism is in LD with the protective allele of the other, in which case the genetic effect of individual SNPs will be blunted by an increase in the frequency of haplotypes that carry 1 predisposing and 1 protective allele [42]. In this study, apparently none of the CTLA4 haplotypes (frequency ⬎5%) from the haplotype blocks were associated with LF infection. This finding might suggest that CTLA4 haplotypes may not be important determinants of parasitic infection. Many studies have previously reported that some haplotype combinations in the CTLA4 polymorphisms were associated with infectious diseases. Analysis of individual haplotypes from HBV-infected subjects revealed that the CTLA4 wildtype haplotype, which included SNPs rs733618, rs5742909, and rs231775, was associated with viral persistence and clearance [36]. Similarly, the haplotype containing the rs733618 and rs231775 major alleles was reported to be strongly associated with high opacity in severe pulmonary tuberculosis [43]. Conflicting results in haplotype association studies of CTLA4 polymorphisms with LF most likely occurred because each study involved only a few hundred samples. A large-scale study of CTLA4 polymorphisms in 8 continental regions by RamÎrez-Soriano et al. indicated that the diversity pattern in the CTLA4 SNP, haplotype frequencies, and LD patterns was higher among populations in Africa than elsewhere [44]. In contrast to population processes that affect the whole genome, gene factors such as differential selection can shape the haplotype structure and LD in specific gene regions and might result in population differences [44]. Because CTLA4 has a key role in the immune system and has been related to autoimmune diseases, the exposure to geographic differential selective pressures, such as the presence of pathogenic infections that could have affected the CTLA4 gene structure, could be envisaged. In summary, the present study has recorded for the first time a significant association of CTLA4 gene polymorphisms with LF and reveals that SNPs rs733618 and rs231775 could be important players in susceptibility to helminth infection in the East Malaysian population. This genetic study may open a new avenue for other studies seeking clinical intervention to target Treg activities in chronic helminth infection in other ethnic populations.
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Acknowledgments We thank the patients and families who participated in this study. This study was supported by grant research from the European Commission TRANCHI (INCO-CT-2006-032436). The first author was financially supported by a National Science Fellowship (NSF) from the Ministry of Science, Technology, and Innovation, Malaysia. References [1] Weil GJ, Ramzy RMR. Diagnostic tools for filariasis elimination programs. Trends Parasitol 2007;23:78 – 82. [2] Michael E. The population dynamics and epidemiology of lymphatic filariasis. In Nutman TB (ed): Lymphatic Filariasis. London, Imperial College Press, 2000, pp 41– 81. [3] Taylor MD, LeGoff L, Harris A, Malone E, Allen JE, Maizel RM. Removal of regulatory T cell activity reverses hyporesponsiveness and leads to filarial parasite clearance in vivo. J Immunol 2005;174:4942–33. [4] Simonsen P, Pedersen E, Lemnge M, Michael E. Lymphatic filariasis research and control in Africa. Parasitol Today 1997;13:413–5. 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Contents lists available at ScienceDirect
Association of CTLA4 gene polymorphisms with lymphatic filariasis in an East Malaysian population Zulkarnain Md Idris a, Noorizan Miswan a, Jamail Muhi b, Tajul Ariffin Awang Mohd c, Ju¨rgen F.J. Kun d, Rahmah Noordin a,* a
Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang, Malaysia Sarawak Health Department, Kuching, Sarawak, Malaysia c Sandakan Health Office, Sandakan, Sabah, Malaysia d Institute for Tropical Medicine, University of Tu ¨ bingen, Tu ¨ bingen, Germany b
A R T I C L E
I N F O
Article history: Received 2 November 2010 Accepted 31 March 2011 Available online 8 April 2011
Keywords: CTLA4 gene Lymphatic filariasis Polymorphisms
A B S T R A C T
Lymphatic filariasis (LF) is a parasitic disease caused by threadlike worms of the Brugia and Wuchereria species that live in the human lymphatic system. Regulatory T cells (Tregs) may play a key role in the pathogenesis of LF, and cytotoxic T-lymphocyte antigen-4 (CTLA4) expressed by Tregs is a potential candidate gene because it modulates T-cell activation. A case– control study was performed to establish a potential association of 5 CTLA4 gene promoter single nucleotide polymorphisms (SNPs; rs733618, rs11571316, rs5742909, rs231775, and rs16840252) with the occurrence of LF in an East Malaysian population (320 LF-infected individuals and 150 healthy controls). Polymorphisms were evaluated using TaqMan real-time polymerase chain reaction followed by direct sequencing. LF carriers of the rs733618 AG genotypes (p ⫽ 0.02) and those with combined minor allele G carriers (AG ⫹ GG; p ⫽ 0.01) exhibited a significantly decreased risk for LF. Among the asymptomatic amicrofilaremic cases, positive associations were reported for all genotypes and variants of rs733618 with odds ratios (ORs) ranging from 0.27 to 0.45. In the asymptomatic microfilaremic cases, marker rs231775 exhibited a significant decreased risk, with ORs ranging from 0.50 to 0.57. The study has identified SNPs in the CTLA4 promoter gene that may be functionally linked with susceptibility to LF. 䉷 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction Lymphatic filariasis (LF) is caused by the vector-borne helminth parasites Wuchereria bancrofti, Brugia malayi, and Brugia timori and is transmitted by mosquitoes [1]. Ninety percent of LF is caused by W bancrofti, which occurs throughout the tropics and subtropics. B malayi infects about 13 million people in south and southeast Asia, whereas B timori is restricted to Timor Leste and a few islands in Indonesia [2]. Adult worms reside in the lymphatics and lymph nodes and induce changes that result in dilatation of lymphatics and thickening of the lymphatic vessel walls. Progressive lymphatic damage and pathology result from the summation of the effect of tissue alterations induced by both living and nonliving adult parasites, the host inflammatory response to the parasites and their secreted antigens, and those seen because of secondary bacterial or fungal infections [3]. A total of 44 million people suffer from 1 or more of the overt manifestations of the infection: lymphedema and elephantiasis of the limbs or genitals, hydrocele, chyluria, pneumonitis, or recurrent infections associated with damaged lymphatics.
* Corresponding author. E-mail address: [email protected] (R. Noordin).
In addition, a significant percentage of infected individuals are outwardly asymptomatic and have no circulating microfilaria but have serologic and radiological evidence of the so-called “cryptic infection” [4,5]. Filarial infections characteristically are associated with a profound downregulated T-cell response to parasite antigen in infected individuals compared with filaria-exposed but uninfected individuals [6]. In essence, some cells of the immune system act in a suppressive or downregulatory manner to switch off inflammatory and protective immune responses mediated by macrophages and eosinophils. These cells, termed regulatory T cells (Tregs), were first defined as inhibiting autoimmune reactions, and such cells appeared to develop naturally in both humans and mice [7]. The regulatory cells release soluble suppressive cytokines (interleukin [IL]-10 and transforming growth factor ), which bind receptors on effector lymphocytes with the effect of deactivating immune cells. The regulatory cells also express a surface molecule, cytotoxic Tlymphocyte antigen-4 (CTLA4) protein, coded by the CTLA4 (CD152) gene, which is stored in endosomal compartments and expressed on the cell surface following activation [8] and delivers an inhibitory signal to T cells in contrast to its homologue CD28. In humans, filarial infections (LF and onchocerciasis) have long been associated
0198-8859/11/$32.00 - see front matter 䉷 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2011.03.017
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with IL-10 and transforming growth factor  and regulatory phenotype T-cell clones have been isolated from onchocerciasis patients [9]. In human LF, infected patients exhibit higher levels of CTLA4 expression in peripheral blood T cells than do uninfected people, and in vitro IL-5 responses (associated with protection) are significantly enhanced in the presence of anti-CTLA4 antibody [10]. Human CTLA4 is encoded by a gene in chromosome 2p33 in a region also containing inducible T-cell costimulator and CD28 genes and shares with the latter a high nucleotide identity, strongly suggesting that they are the result of a gene duplication [11]. Many single nucleotide polymorphisms (SNPs) have been identified in the CTLA4 region [12,13]. SNPs in the promoter region can affect the binding affinity of transcriptional factor involved in the regulation of gene expression [14]. We hypothesized that potentially functional CTLA4 promoter SNPs and haplotypes could alter transcriptional activity, thus affecting susceptibility to develop LF, and might explain some of the between-person differences in LF manifestations. To date, no study has comprehensively investigated the CTLA4 promoter SNPs on their functional roles and contributions to the risk of developing LF. To test our hypothesis, polymorphisms located in the CTLA4 promoter were identified from the public dbSNP (http:// www.ncbi.nlm.nih.gov/projects/SNP/) database. Four promoter polymorphisms, rs733618 (⫺1,722 A/G), rs11571316 (⫺1,577 C/T), rs5742909 (⫺319 C/T), and rs16840252 (⫺1,477 C/T), and exon 1 SNP (i.e., rs231775 [⫹48 A/G]) were studied by allelic discrimination genotyping assay. 2. Subjects and methods The study protocol complied with the Declaration of Helsinki and was approved by the Committee on the Ethics of Human Research of Universiti Sains Malaysia. Written informed consent was obtained from each participant. 2.1. Subjects The study population comprised a total of 320 individuals (179 males and 141 females) from LF-endemic areas. The ages ranged from 3 to 78 years (28.23 ⫾ 19.68 years) and subjects were recruited from various districts from the state of Sarawak between September 2004 until March 2006 and the state of Sabah in December 2008. Nocturnal subperiodic B malayi is the species prevalent in these areas. Three rounds of mass drug administration (MDA) have been performed in this area with pre-MDA microfilaria (mf) prevalence of 2% (20/1,000 population). The mf prevalence at the time of the study was 2.64% (32/1,211) from 16 villages; this unchanged mf prevalence rate was probably caused by treatment noncompliance or insufficient MDA coverage because of the logistic challenges in this area. A resident of the endemic area was defined as someone who has lived in the community for at least 10 years and who has not been absent for more than 6 months. Children younger than 10 years were confirmed as residents who have lived in the community since birth. The age prevalence curve increased from under 3% in the age group 3 to 5 years to near 30% between ages 11 and 20 and then declined after the age of 20. The prevalence among middle-age adults was less than 20%, whereas the prevalence between genders was not significantly different.
All individuals are native people of East Malaysia, with the majority of Malay ethnicity, and some are indigenous people of Sabah (Kadazan and Bajau) and Sarawak (Iban and Melanau). However, these ethnic differences are not considered significant. For all patients with LF, the diagnostic test for LF infection was performed using the traditional thick blood smear and pooled real-time polymerase chain reaction (PCR; pooled blood spot samples from 10 individuals per pool) for the presence of mf and BRUGIArapid. The rapid test uses BmR1 recombinant antigen that detects specific antifilarial immunoglobulin (Ig)-G4 antibodies in individuals infected with B malayi and B timori [15–17]. Elevated levels of antifilarial IgG4 antibodies are a marker of active LF [18 –20]. The screening was based on household survey. In the morning, houseto-house visits were made. The villagers were given a personal tag on their wrists and were asked about their personal information (demographic data), drug (diethylcarbamazine ⫹ albendazole) intake, bednet use, and symptoms. The location of each household was recorded by a global positioning system. Villagers were then asked to gather at a community hall or village head’s house at 8 PM for sample collection. A custom-made barcoding system was used to label all wrist tags, blood tubes, slides, rapid test cassettes, and aliquots. The individuals with LF were divided into 2 groups, namely, asymptomatic microfilaremia (AM; i.e., AM/mf⫹/IgG4⫹) and asymptomatic amicrofilaraemia (AsAm; i.e., AsAm/mf⫺/ IgG4⫹). Asymptomatic individuals were assessed based on an examination of their limbs to look for symptoms of edema (limb enlargement) and direct questions regarding LF symptoms (edema, lymphangitis, fever) by field staff who spoke in the local dialect. The AM group consisted of 109 individuals (26.01 ⫾ 20.02 years) and was characterized by the presence of mf in 60 l of finger-prick blood and positive result by the rapid test. The AsAm group comprised 211 individuals (29.28 ⫾ 19.48 years) who were negative by thick blood smear, positive by the rapid test, and negative by the pooled real-time PCR. The healthy endemic control group of uninfected people consisted of 150 individuals (64 males and 86 females) aged 3 to 70 years (21.3 ⫾ 16.29 years) who were negative by all diagnostic methods and were selected from community volunteers from the same district. 2.2. SNP selection and genotyping The identification of SNPs associated with LF was performed by examining the relationship of promoter SNPs of the CTLA4 gene to LF. Using public databases, including PubMed (National Center for Biotechnology Information) and Online Mendelian Inheritance in Man, 5 SNPs were selected. Based on the previous reports, these SNPs may be expected to result in a change of function or expression of the encoded protein [13,21]. Genomic DNA was extracted from blood spots of each participant using a QIAamp mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. All SNPs were genotyped using a TaqMan assay in a 72-well rotor format (Corbett Research, New South Wales, Australia). All probes and primers were designed by the Assay-by-Demand service from Applied Biosystems (Foster City, CA; Table 1). Genotyping was performed in 20-l reactions comprising 10 ng of genomic DNA, 1⫻ TaqMan SNP genotyping assay (consisting of 200 nM TaqMan MGB probes, FAM and VIC
Table 1 Positions, nucleotide variations, and assay ID for CTLA4 DbSNP ID
Chromosome position
Relative position/region
Allele
Assay ID (Applied Biosystems)
rs733618 rs11571316 rs16840252 rs5742909 rs231775
Chr2q33: 204439189 Chr2q33: 204439334 Chr2q33: 204439764 Chr2q33: 204440592 Chr2q33: 204440959
⫺1,722/promoter ⫺1,577/promoter ⫺1,147/promoter ⫺319/promoter ⫹48/exon 1 (17 T/A)
A/G C/T C/T C/T A/G
C_2415791_10 C_30981395_10 C_32900355_10 C_27834180_10 C_2415786_20
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dye-labeled, and 900 nM of primers), and 2⫻ HotStarTaq Master Mix (containing 1.5 mM MgCl2, 200 M dNTPs, HotStarTaq DNA polymerase, and RNase-free water; Qiagen, Hilden, Germany). Real-time PCR reactions were thermally cycled with an initial denaturation step at 95⬚C for 15 minutes, followed by 40 cycles of 94⬚C for 30 seconds, 60⬚C for 1 minute, and 72⬚C for 1 minute and then a final extension step at 72⬚C for 10 minutes. Real-time fluorescence detection was performed during the annealing/extension step of each cycle. To ensure genotyping quality, positive control and nontemplate negative controls were included for each genotype in each run. Allelic discrimination was performed on a Rotor-Gene 6000 Multiplex System and data analysis was conducted with the software interface version 1.7. Subsequently, randomly selected samples were genotyped through direct sequencing for genotype confirmation and 100% concordance was obtained for every condition. Nucleotide changes were detected by visual inspection of chromatograms. 2.3. Statistical analysis Statistical analysis was performed using PASW Statistic software 17.0 (SPSS, Inc., Chicago, IL). Allele and genotype frequencies were calculated on LF and control subjects by direct counting. The CTLA4 SNPs were tested for Hardy–Weinberg equilibrium (HWE) by comparing observed and expected genotype frequencies in both LF and control groups using the 2 test (p value ⬎ 0.05 was considered consistent with HWE). The associations of polymorphisms were estimated by logistic regressions analysis with odds ratios (OR) and 95% confidence intervals (95% CI). The level of statistical significance was set at a p value less than 0.05 (2-tailed test). For multiple marker association analyses, the estimation of pairwise linkage disequilibrium (LD) values and haplotype block structure was performed using the default method of Gabriel et al. [22] and displayed schematically with their population frequencies and interblock connections based on the Haploview program output. Haplotype analysis was performed using SNPAnalyzer version 2.0 (http:// snp.istech21.com/snpanalyzer/2.0/).
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3. Results Five SNPs (i.e., rs733618, rs11571316, rs16840252, rs5742909, and rs231775) in the CTLA4 gene promoter and exon 1 region were genotyped in 320 individuals (109 AM and 211 AsAM) with LF, as well as in 150 endemic control subjects. The frequency of the genotypes in the LF group followed HWE. Two markers, namely, rs733618 and rs11571316, demonstrated borderline significant deviation (p ⫽ 0.01– 0.05) from HWE (underlined in Table 2), but no marker had an HWE p value less than 0.05 in more than 1 dataset, so all markers were used for the association analysis. Individual SNPbased association analysis revealed a number of significant associations between several SNPs and disease manifestations in the LF samples (Table 2). Logistic regression analysis demonstrated that the LF carriers of the rs733618 AG genotypes and those with combined minor allele G carriers (AG ⫹ GG) exhibited a significant decrease risk for LF (OR ⫽ 0.61, p ⫽ 0.02, 95% CI ⫽ 0.41– 0.91; and OR ⫽ 0.61, p ⫽ 0.01, 95% CI ⫽ 0.41– 0.90, respectively). No significant relationships between any other SNPs were observed. However, among the AsAm group, positive associations with disease were observed for all genotypes and variants of rs733618, with ORs ranging from 0.27 (95% CI ⫽ 0.09 – 0.84, p ⫽ 0.02, for the GG genotype) to 0.45 (95% CI ⫽ 0.29 – 0.70, p ⫽ 0.001, for the AG genotype). In the AM group, marker rs231775 exhibited a significant decrease in risk and directly correlated with disease manifestation, with ORs ranging from 0.50 (95% CI ⫽ 0.15– 0.75, p ⫽ 0.008, for the GG genotype) to 0.57 (95% CI ⫽ 0.33– 0.97, p ⫽ 0.04, for the AG genotype). No significant associations were observed for the other SNPs in the AM cases. Four SNPs, namely, rs733618, rs11571316, rs16840252, and rs5742909, within the LF haplotype block gave 9 different haplotype combinations (Table 3). Four haplotypes gave more than 5% in frequencies, ATCC, GCCC, ACCC, and ACTT, and at least 3 major alleles were found in each genotype. The ATCC haplotype demonstrated the highest frequencies, with 39.7%. In LF disease manifestations, both SNPs rs11571316 and rs16840252 were defined in the
Table 2 Association of CTLA4 polymorphisms with LF and disease manifestation in the East Malaysian population SNP
rs733618
HWE rs11571316
HWE rs16841252
HWE rs5742909
HWE rs231775
HWE
Genotype
AA AG GG AG ⫹ GG CC CT TT CT ⫹ TT CC CT TT CT ⫹ TT CC CT TT CT ⫹ TT AA AG GG AG ⫹ GG
Control n (%)
66 (44.0) 75 (50.0) 9 (6.0) 84 (56.0) 0.04 48 (32.0) 85 (56.7) 17 (11.3) 102 (68.0) 0.02 121 (80.7) 29 (19.3) 0 (0.0) 29 (19.3) 0.19 121 (80.7) 29 (19.30 0 (0.0) 29 (19.3) 0.19 52 (34.6) 70 (46.7) 28 (18.7) 98 (65.3) 0.61
LF (AM and AsAsm)
AM
n (%)
OR
95% CI
p value
180 (56.3) 125 (39.0) 15 (4.7) 140 (43.8)
1.00 (RV) 0.61 0.61 0.61
0.41–0.91 0.26–1.47 0.41–0.90
0.02 0.27 0.01 0.25
106 (33.1) 167 (52.2) 47 (14.7) 214 (66.9)
1.00 (RV) 0.89 1.25 0.95
0.58–1.37 0.65–2.40 0.63–1.44
0.59 0.50 0.81 0.15
247 (77.2) 69 (21.6) 4 (1.2) 73 (22.8)
1.00 (RV) 1.17 — 1.23
0.72–1.89 — 0.76–2.00
0.54 — 0.39 0.65
251 (78.4) 64 (20.0) 5 (1.6) 69 (21.7)
1.00 (RV) 1.06 — 1.14
0.65–1.74 — 0.71–1.86
0.80 — 0.58 0.69
128 (40.0) 145 (45.3) 47 (14.7) 192 (60.0)
1.00 (RV) 0.84 0.68 0.80
0.55–1.29 0.39–1.20 0.53–1.19
0.43 0.19 0.27 0.57
AsAm
n (%)
OR
95% CI
p value
44 (40.4) 55 (50.5) 10 (9.1) 65 (59.6)
1.00 (RV) 1.10 1.67 1.16
0.67–1.84 0.63–4.43 0.70–1.91
0.72 0.31 0.56 0.22
43 (39.4) 57 (52.3) 9 (8.3) 66 (60.6)
1.00 (RV) 0.75 0.59 0.72
0.44–1.27 0.24–1.46 0.43–1.21
0.29 0.26 0.22 0.10
93 (85.3) 15 (13.9) 1 (1.0) 16 (14.7)
1.00 (RV) 0.63 — 0.72
0.34–1.33 — 0.37–1.34
0.25 — 0.33 0.10
93 (85.3) 14 (12.8) 2 (1.8) 16 (14.7)
1.00 (RV) 0.63 — 0.72
0.32–1.26 — 0.37–1.40
0.19 — 0.33 0.11
56 (51.4) 43 (39.4) 10 (9.2) 53 (48.6)
1.00 (RV) 0.57 0.33 0.50
0.33–0.97 0.15–0.75 0.30–0.83
0.04 0.008 0.007 0.68
n (%)
OR
95% CI
p value
136 (64.5) 70 (33.2) 5 (2.3) 75 (35.5)
1.00 (RV) 0.45 0.27 0.43
0.29–0.70 0.09–0.84 0.28–0.67
0.001 0.02 0.001 0.25
63 (29.9) 110 (52.1) 38 (18.0) 148 (70.1)
1.00 (RV) 0.99 1.70 1.11
0.62–1.58 0.86–3.38 0.70–1.74
0.95 0.13 0.66 0.40
154 (73.0) 54 (25.6) 3 (1.4) 57 (27.0)
1.00 (RV) 1.46 — 1.54
0.88–2.43 — 0.93–2.56
0.14 — 0.09 0.48
158 (74.9) 50 (23.7) 3 (1.4) 53 (25.1)
1.00 (RV) 1.32 — 1.40
0.79–2.21 — 0.94–2.33
0.29 — 0.20 0.67
72 (34.1) 102 (48.3) 37 (17.5) 139 (65.9)
1.00 (RV) 1.05 0.95 1.02
0.66–1.68 0.52–1.75 0.66–1.60
0.83 0.88 0.92 0.93
AM ⫽ asymptomatic microfilaremic; AsAm ⫽ asymptomatic amicrofilaremic; 95% CI ⫽ 95% confidence interval; OR ⫽ odds ratio; RV ⫽ reference value; HWE ⫽ Hardy–Weinberg equilibrium.
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Table 3 Haplotype frequencies in an LF population Haplotype frequencies Block 1 LF SNP Allele Hap 1 Hap 2 Hap 3 Hap 4 Hap 5 Hap 6 Hap 7 Hap 8 Hap 9 Block 1 AM SNP Allele Hap 1 Hap 2 Hap 3 Block 1 AsAm SNP Allele Hap 1 Hap 2 Hap 3 Hap 4 Hap 5
rs733618 A/G A G A A A G A G G
rs11571316 C/T T C C C C T C T C
rs11571316 C/T C T C
rs16840252 C/T C C T
rs11571316 C/T T C C C C
rs16840252 C/T C C T T C
rs16840252 C/T C C C T T C C C T
rs5742909 C/T C C C T C T T C C
0.397 0.255 0.223 0.104 0.007 0.003 0.003 0.003 0.001
0.547 0.344 0.078 rs5742909 C/T C C T C T
0.441 0.412 0.130 0.012 0.005
AM ⫽ asymptomatic microfilaremic; AsAm ⫽ asymptomatic amicrofilaremic; LF ⫽ lymphatic filariasis.
same haplotype blocks for AM and AsAm, whereas an additional SNP, rs5742909, was reported only in the AsAm haplotype block. All haplotypes (3 AM haplotypes and 5 AsAm haplotypes) were at least or more than 5%, and the CC and TCC haplotype gave the highest frequency distribution, with 54.7 and 44.1% in AM and AsAm, respectively. 4. Discussion Commonly known as elephantiasis, LF is a devastating parasitic infection transmitted by mosquitoes. Less than half of the infected people are clinically symptomatic, yet all have hidden damage to their lymphatic and/or renal systems. This state of asymptomatic microfilaremia is associated with a highly downregulated immune system [23], but it is unclear how, when, or even whether these individuals will progress to develop 1 or more overt clinical manifestations of filarial disease. With the circulating mf in their blood, they are an important source of infection. A second asymptomatic presentation is termed AsAm. In the endemic areas, a proportion of the population does not exhibit mf or clinical manifestation although they are exposed to infected larva at a rate similar to that of individuals who became microfilaremic. Their infections are defined not by microfilaremia, but by the presence of parasite antigen or antifilarial IgG4 antibodies in the blood. CTLA4 plays an essential role in immunologic homeostasis as a negative regulator of T-cell activation. It is highly expressed by Tregs and could play an important role in their function [24]. A number of studies have demonstrated that polymorphisms, especially in the CTLA4 gene promoter region, are associated with autoimmune, inflammatory, and noninfectious diseases such as ankylosing spondylitis [25–29]. However, such an association, especially in promoter polymorphisms of the CTLA4 gene in helminth human infections, has not been well investigated. Our study demonstrated that rs231775 (⫹49) minor allele G carriers and genotype among AM individuals exhibited a positive association with LF in the East Malaysian population. Previous studies revealed that the CTLA4 rs231775 (⫹49) variation has a
functional effect on the expression level of CTLA4 [30,31]. The rs231775 (⫹49) G allele has been associated with increased risk for diseases resulting from an overly vigorous immune response, including insulin-dependent diabetes mellitus and autoimmune disorders. Likewise, in an earlier study this allele was also associated with improved clearance of hepatitis C virus infection resulting from interferon ␣– based therapy [32]. These studies could not rule out the role of other SNPs linked to ⫹49 because the other haplotype-tagging SNPs were not studied. The A-to-G mutation at position ⫹49 leads to a nonsynonymous amino acid change from threonine to alanine, thus changing the polarity of the amino acid and potentially altering the function of the protein in the signal peptide. Lymphocytes from donors carrying the rs231775 (⫹49) G allele appeared to express less CTLA4 on their surfaces, proliferate more under conditions of suboptimal activation, and exhibit less CTLA4-mediated inhibition of T-cell responses [30,31]. A study by Anjos et al. indicated that the predisposing Ala allele (⫹49G) is incompletely glycosylated in the endoplasmic reticulum, leading to retrograde transport of a portion of the molecules to cytoplasm for degradation [33]. They also demonstrated that persons with homozygous GG have one-third less CTLA4 on their T-cell surface than people with homozygous AA. This ultimately results in less mature CTLA4 (Ala) at the cell surface, which may explain in part the reduced inhibitory function of CTLA4 reported in individuals with the ⫹49G allele [31]. With regard to infectious disease, a study including 391 individuals in a cohort from Finland demonstrated a significant difference between Helicobacter pylori seropositive and negative individuals in enzyme catalyzed oxidative degradation of tryptophan activity only in rs231775 (⫹49) G allele carriers [34]. This finding also agrees with that of FernÂndez-Mestre et al., who demonstrated that the rs231775 (⫹49) allele and genotype were associated with a risk of parasitic disease [21]. The analysis involved different clinical groups of patients infected with Leishmania and demonstrated an increased frequency of heterozygous AG genotype in patients with diffused cutaneous leishmaniasis compared with patients with localized cutaneous leishmaniasis and intermediate cutaneous leishmaniasis. This indicated that the heterozygous genotype associated with overactivation of T-cell proliferation could confer susceptibility to the development of the more severe clinical form of cutaneous leishmaniasis [21]. In addition, the homozygous GG genotype of rs231775 (⫹49) has been reported to be associated with hepatitis C virus infection [32]. Similarly, the logistic regression model by Hu et al. demonstrated that the CTLA4 rs231775 (⫹49) variant genotype GG was associated with an increased risk for 2 virus infection–related cancers, namely, hepatocellular carcinoma and cervical cancer, by 1.46-fold and 1.55-fold when compared with common homozygous AA [35]. The present study demonstrated that rs733618 (⫺1,722) minor allele carriers and genotypes had a positive association with LF (AM and AsAm), as well as with AsAm, but not with AM. Thus, this may imply that individuals with rs733618 (⫺1,722) SNP may have a predisposition to become AsAm. Thio et al. also suggest a role for the promoter SNP rs 733618 (⫺1,722) in hepatitis B virus (HBV) recovery [36]. Its strong association may be the result of its linkage with the SNP rs231775 (⫹48), because rs733618 (⫺1,722) haplotype combinations were only associated with the G allele of rs231775 (⫹49). Alternatively, because it is located in the promoter region of the gene, it may alter the transcriptional regulation of CTLA4. If rs733618 (⫺1,722) has an independent association, the data would support decreased CTLA4 production with the rs733618C allele, leading to an increased chance of recovery from HBV infection [36]. Previous studies have demonstrated that the functional effect of genetic variation may lead to interindividual differences in immune response capacity [37]. These studies were supported by the
Z.M. Idris et al. / Human Immunology 72 (2011) 607-612
association of several known SNPs in immunomodulatory genes and altered immune capacity [38]. Thus, given that parasite infection may in part depend on escaping immune surveillance, the SNPs rs733618 (⫺1,722) and rs231775 (⫹49) may constitute risk factors for susceptibility to parasitic infections. In the present study, the results obtained by specific antibody and immunomodulatory genes such as CTLA4 (expressed by Treg) may suggest that the interindividual differences contributed to disease susceptibility. In this study, both AM and AsAm types of LF asymptomatic individuals have elevated IgG4 antibodies compared with healthy controls. It has been suggested that the activation of Treg cells in asymptomatic carriers keeps the pathology mild enough to avoid symptoms [39]. Unlike other IgG subclasses, IgG4 is found under conditions where IL-10 produced by Tregs is increased after chronic helminth infections [40]. Furthermore, a recent study reported that an intermediate degree of correlation indicated the induction of IgG4 antibody also involved cell– cell contact with CTLA4 in CD4⫹CD2⫹ Tregs [9]. In addition, CTLA4 rs231775 (⫹49) has demonstrated an association with autoimmune pancreatitis, which is characterized serologically by an increased IgG4 level [41]. One haplotype block was identified for each cohort from the LD structure. Associated SNP rs733618 was found to be located in the block structure of the LF cohort but none of the associated SNPs (rs733618 and rs231775) was found in the block structure when LF types were computed. Interestingly, consistently strong LD (D= and r2) was reported between rs16840252 and rs5742909 across all LD structures (data not shown). In the presence of 2 functional polymorphisms on the same LD block, it is possible that the predisposing allele of 1 polymorphism is in LD with the protective allele of the other, in which case the genetic effect of individual SNPs will be blunted by an increase in the frequency of haplotypes that carry 1 predisposing and 1 protective allele [42]. In this study, apparently none of the CTLA4 haplotypes (frequency ⬎5%) from the haplotype blocks were associated with LF infection. This finding might suggest that CTLA4 haplotypes may not be important determinants of parasitic infection. Many studies have previously reported that some haplotype combinations in the CTLA4 polymorphisms were associated with infectious diseases. Analysis of individual haplotypes from HBV-infected subjects revealed that the CTLA4 wildtype haplotype, which included SNPs rs733618, rs5742909, and rs231775, was associated with viral persistence and clearance [36]. Similarly, the haplotype containing the rs733618 and rs231775 major alleles was reported to be strongly associated with high opacity in severe pulmonary tuberculosis [43]. Conflicting results in haplotype association studies of CTLA4 polymorphisms with LF most likely occurred because each study involved only a few hundred samples. A large-scale study of CTLA4 polymorphisms in 8 continental regions by RamÎrez-Soriano et al. indicated that the diversity pattern in the CTLA4 SNP, haplotype frequencies, and LD patterns was higher among populations in Africa than elsewhere [44]. In contrast to population processes that affect the whole genome, gene factors such as differential selection can shape the haplotype structure and LD in specific gene regions and might result in population differences [44]. Because CTLA4 has a key role in the immune system and has been related to autoimmune diseases, the exposure to geographic differential selective pressures, such as the presence of pathogenic infections that could have affected the CTLA4 gene structure, could be envisaged. In summary, the present study has recorded for the first time a significant association of CTLA4 gene polymorphisms with LF and reveals that SNPs rs733618 and rs231775 could be important players in susceptibility to helminth infection in the East Malaysian population. This genetic study may open a new avenue for other studies seeking clinical intervention to target Treg activities in chronic helminth infection in other ethnic populations.
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Acknowledgments We thank the patients and families who participated in this study. This study was supported by grant research from the European Commission TRANCHI (INCO-CT-2006-032436). The first author was financially supported by a National Science Fellowship (NSF) from the Ministry of Science, Technology, and Innovation, Malaysia. References [1] Weil GJ, Ramzy RMR. Diagnostic tools for filariasis elimination programs. Trends Parasitol 2007;23:78 – 82. [2] Michael E. The population dynamics and epidemiology of lymphatic filariasis. In Nutman TB (ed): Lymphatic Filariasis. London, Imperial College Press, 2000, pp 41– 81. [3] Taylor MD, LeGoff L, Harris A, Malone E, Allen JE, Maizel RM. Removal of regulatory T cell activity reverses hyporesponsiveness and leads to filarial parasite clearance in vivo. J Immunol 2005;174:4942–33. [4] Simonsen P, Pedersen E, Lemnge M, Michael E. Lymphatic filariasis research and control in Africa. Parasitol Today 1997;13:413–5. 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