Background
Chagas disease (American trypanosomiasis) is caused by the protozoan Trypanosoma cruzi and transmitted by the reduviid bug. It occurs exclusively in the Americas, particularly in poor, rural areas of Mexico, Central America, and South America. The disease remains endemic in Latine America where the vector-based transmission is still active in some countries. Imported disease is increasingly recognized as an emerging problem in the USA and Europe due to immigration from Latin America. It is estimated that as many as 8–9 million people have Chagas disease. Approximately, 40 million people are currently at risk of infection
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. Decades after acute infection, approximately 30% of infected individuals develop Chronic Chagas cardiomyopathy (CCC), one of the most important consequence of T. cruzi infection. CCC is an inflammatory dilated cardiomyopathy, with a potentially fatal outcome. 5 to 10% of infected individuals develop digestive disease. The remaining two-thirds of infected individuals remain asymptomatic (ASY) and free from heart disorders for life
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. 20,000 deaths attributable to Chagas disease occur annually, typically due to CCC
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. Heart failure due to CCC has a worse prognosis with 50% shorter survival when compared to other cardiomyopathies of different etiologies
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5
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The dynamics of the immune response to T. cruzi is that of a persistent infection with an obligatory intracellular parasite. During acute T. cruzi infection, T. cruzi pathogen-associated molecular patterns (PAMPs) trigger innate immunity in multiple cell types
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, which release proinflammatory cytokines and chemokines, such as IL-1, IL-6, IL-12, IL-18, TNF-α, CCL2, CCL5, and CXCL9 activating and mobilizing migration of cascades of inflammatory cells
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. Antigen-presenting cells subsequently elicit a strong T cell and antibody response against T. cruzi, where IL-12 and IL-18 drive the differentiation of IFN-γ-producing T. cruzi–specific Th1 T cells which migrate to sites of T. cruzi-induced inflammation, including the myocardium, in response to locally produced chemokines
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. Th1 T cell and antibody responses lead to control but not complete elimination of tissue and blood parasitism, establishing a low-grade chronic persistent infection by T. cruzi. As a result of persistent infection, both CCC and ASY chronic Chagas disease patients show a skewed Th1-type immune response
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, but those who develop Chagas cardiomyopathy display a particularly strong Th1-type immune response with increased numbers of IFN-γ-producing T cells in peripheral blood mononuclear cells (PBMC)
13
as well as plasma TNF-α in comparison with uninfected or ASY patients
14
. PBMC of CCC patients also display increased levels of IFN-γ- or TNF-α producing CCR5/CXCR3+ CD4+ T cells
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. In addition, CCC patients display a reduced number of CD4+CD25highIL-10+ and CD4+CD25highFoxP3+ regulatory T cells in their peripheral blood as compared to patients in the ASY form of Chagas disease, suggesting such cells may play a role in the control of the intensity of inflammation in chronic Chagas disease
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. Furthermore, PBMC from CCC patients displayed increased numbers of CD4+CD25highFoxP3+CTLA-4+ T cells, and decreased numbers of as compared to ASY patients. These reports suggest that a smaller CD4+FoxP3+/CD25+ Treg compartment with deficient suppressive activity exists in CCC patients, leading to uncontrolled production of Th1 cytokines
18
. Circulating CD4+IL-17+ T cells appear in low frequency in PBMC from CCC patients as compared with ASY patients and non-infected individuals
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. On the whole, these results suggest that proinflammatory cells and cytokines are markers associated with progression to CCC, whereas the production of IL-10, IL-17 and increased numbers of regulatory T cells are markers of protection from CCC development, indicating that failure to regulate Th1 responses may be the underlying immune defect of patients who progress to CCC.
The exacerbated Th1 response observed in the PBMC of CCC patients is reflected on the Th1-rich myocardial inflammatory infiltrate, with mononuclear cells predominantly producing IFN-γ and TNF-α, with lower production of IL-4, IL-6, IL-7, and IL-15
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. It has recently been shown that CCL5+, CCXCL9+, CCR5+, CXCR3+ cells were abundant in CCC myocardium, and mRNA levels of the Th1-chemoattracting chemokines CXCL9, CXCL10, CCL2 (also known as MCP-1), CCL3, CCL4, CCL5; along with CCL17, CCL19, CCL21 and their receptors were also found to be upregulated in CCC heart tissue
12
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. Importantly, median expression of CCL5, a CCR5 ligand, was the highest among all chemokines tested (166-fold increase over control). Significantly, the intensity of the myocardial infiltrate was positively correlated with CXCL9 mRNA expression. Moreover, a single nucleotide polymorphism in the CXCL9 gene, associated with a reduced risk of developing severe CCC in a cohort study, was associated with reduced CXCL9 expression and intensity of myocarditis in CCC
22
. These results are consistent with a major role of locally produced Th1-chemoattractant chemokines in the accumulation of CXCR3/CCR5+ Th1 T cells in CCC heart tissue
23
.
Familial aggregation of CCC has been described, suggesting that there might be a genetic component to disease susceptibility
24
. Several genes were associated to an increased risk to develop cardiomyopathy (HLA, MHC, TNF, IL1A, IL1B, IL1RN, IL10, IL12B, TIRAP, CCL2, BAT1, LTA, IKBL, CCR5, MIF, IFNG, CXCL9, CXCL10)
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. So far, up to 30 case control studies were done (see for review
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). These studies often led to inconclusive results that may be explained in different ways: a) the use of seronegative subjects as controls which are inadequate controls, since it is unknown whether they were exposed to the pathogen; b) the relatively small size of the study groups which affected the power (the probability) to detect an association; c) the number of tested SNPs; d) the highly heterogeneous genetic background of the study population due to admixture; e) the sex ratio known to exist has not been taken in consideration
54
.
Among these susceptibility studies, putative implication of genes crucially involved in the innate immunity-such as the Toll like receptors (TLR) and some of its most relevant signalling molecules like TIRAP was searched for. Two studies on the TLR and TIRAP failed to identify disease associations with TLR 1,2, 5, 6 and 9; in one of the reports an association was found with a TLR4 SNP among Chilean chagasic patients
55
, while in the second study – which enrolled nearly double the number of Brazilian Chagasic individuals - no association was found with TLR4, but instead with TIRAP S180L heterozygosity
41
. Chemokines are key players in controlling migration of specific cell types bearing their receptors to sites of tissue inflammation, and associations between CCR5 –involved in T cell and macrophage migration and CCL2 –involved in monocyte migration - with CCC were reported
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. Both processes, TLR signaling and chemokine-mediated cell migration are of paramount importance in Chagas disease and are key to the pathogenesis of CCC. Here, we conducted a study focusing on TIRAP, CCL2 and CCL5. Thorough genetic analysis, testing multiple tag SNPs per gene and thus detecting any possible relevant genetic variants in a large Brazilian population and ASY subjects as controls we could have a sensitive assessment of the contribution of genetic variants in prognosis to CCC either confirming or finding additional associated SNPs in the mentioned genes. This can be considered a candidate gene replication study, performed with a larger cohort of Chagas patients and only comparing CCC to the asymptomatic seropositive (ASY) patient group. Significant associations were found for CCR5, CCL2, and TIRAP genes.
Methods
Ethical standard
Written informed consent was obtained from all the patients, in accordance with the guidelines of the various internal review boards of all the involved institutions. The protocol was also approved by the INSERM Internal Review Board and the Brazilian National Ethics in Research Commission (CONEP). All the patients enrolled in this study were over 21 years old so paternal consent was not required. In the case of samples from heart donors, written informed consent was obtained from their families. Investigations were conformed to the principles outlined in the declaration of Helsinki.
Diagnostic criteria
The diagnostic criteria for Chagas disease included the detection of antibodies against T. cruzi in at least two of three independent serological tests (EIA [Hemobio Chagas; Embrabio São Paulo], indirect immunofluorescence assays [IFA-immunocruzi; Biolab Merieux], and indirect hemagglutination tests [Biolab Merieux])
12
. All Chagas disease patients underwent standard electrocardiography and echocardiography. Echocardiography was performed at the hospital, with a Sequoia model 512 echocardiograph with a broad-band transducer. Left ventricular dimensions and regional and global function, including the recording of left ventricular ejection fraction (LVEF), were evaluated with a two-dimensional, M-mode approach, in accordance with the recommendations of the American Society of Echocardiography. ASY subjects had no electrocardiography and echocardiography changes. CCC patients presented typical conduction abnormalities (right bundle branch block and/or left anterior division hemiblock)
56
. CCC patients with significant left ventricular systolic dysfunction (LVEF <40%) were classified as having severe CCC, whereas those with no significant ventricular dysfunction (LVEF ≥40%) were classified as having moderate CCC. We selected 40% as arbitrary cutoff value that has been previously used to define significant ventricular dysfunction by our group and others
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58
.
Study population for polymorphism analysis
The patients and ASY controls were born and raised in rural areas of Sao Paulo, Minas Gerais and Bahia states and enrolled in one of the study centers (Incor, FMUSP, FMRP, UFTM, IDPC). Patients with digestive forms were excluded of this study. Patients were classified as ASY (n = 118) or as having CCC (n = 315). ASY individuals were used as the control subjects for this study because they were from the same areas of endemicity as the patients with CCC, had encountered the parasite and had tested seropositive for T. cruzi infection, but the infection had not progressed to CCC. Of the 118 ASY subjects, 45.3% were male, whereas in the CCC patients group, this percentage reaches 61.3%. The difference in sex distribution between the groups was significant (p = 1.21E-4; OR = 2.126; 95% CI: 1.450 – 3.12). It is well known that male patients infected with T. cruzi have a higher risk of progression to CCC than female patients
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. Of 315 patients with CCC, 106 (42 men [39.6%] and 64 women [60.4%]) showed no significant ventricular dysfunction and were thus classified as having moderate CCC, whereas 199 (144 men [72.4%] and 55 women [27.6%]) had severe ventricular dysfunction and were classified as having severe CCC. Data for left ventricular ejection fraction were missing for 10 patients with CCC. So, when we compared moderate patients to severe patients, these 10 individuals were excluded from the analysis. Regarding progression of the ASY cases to CCC, the yearly progression rate –regardless of age group- is ca. 1-2%/year. The average age of Subjects with asymptomatic form was above 55 years. Taking into account that they were all born in endemic areas before vector transmission was interrupted, it is likely that in most if not all cases vector-borne infection occurred in early childhood. The odds that a significant number of such mature patients convert to CCC, and that this thwarts our statistical calculation is rather low; however, this is a pitfall of all cross sectional studies on diseases that display progression.
Blood samples and DNA preparation
For each subject, 5 to 15 ml of blood were collected in EDTA tubes. Genomic DNA was isolated on a silica-membrane according to the manufacturer’s protocol (QIAamp DNA Blood Max Kit, Qiagen, Hilden, Germany).
SNP selection
Tag single nucleotide polymorphisms (SNPs) were selected on the basis of HapMap Data for the Caucasian and Yoruba reference populations. Tag SNPs were selected within a region extending 5 kb on either side of the candidate gene. The minor allele frequency (MAF) cut off value was arbitrarily set at 20% (so the markers characterized by a MAF < 20% were excluded from the analysis by lack of power). In each reference population, the markers with MAF > 20% are included in different blocks of correlation (based on the r2 values). One marker in each block was selected and considered as a Tag SNPs. Indeed, markers located in the same block of correlation gave the same genetic information in association studies. Tag SNPs characterised by a MAF over 20% on at least one reference population were selected. These Tag SNPs were defined to catch all the genetic information from the candidate gene. We selected three tag SNPs for CCR5, six tag SNPs for CCL2 and six tag SNPs for MAL/TIRAP genes. Taking into account a disease with a prevalence of 30%, a cutoff for significant association of 0.05, for a genotype relative risk of 1.3, the probability to detect a real association reaches 63% with 315 chronic cases and 118 ASY controls. We decided to use a cut off of 20% instead of 10% or 15%. For lower cut off, the number of Tag SNPs will increase and it will request a seriously large your study population to have a good statistical power.
SNP genotyping
Most of the genotyping was done with the Golden Gate genotyping assay (Illumina, San Diego, USA). In some cases, genotyping assays were performed with the TaqMan system (Applied Biosystems, Foster City, USA) according to the manufacturer’s instructions.
Statistical analysis
SPSS Statistics software v. 17.0 (IBM, Armonk, USA) was used for statistical analyses. We performed stepwise binary logistic regression analysis on the whole population, to analyse the relationship between the probability of an individual to develop chronic Chagas cardiomyopathy and the main covariates (sex and polymorphisms). Sex was considered as a binary covariate. In our stepwise binary logistic regression analysis, genotypes were considered as binary covariates. Indeed, for each polymorphism we had two alleles (A frequent one; a rare one). So, we obtained three genotypes (AA, Aa and aa). In our stepwise binary logistic regression analysis, genotypes were considered as binary covariates. So, we performed three different analyses (Analysis 1: AA vs Aa + aa (we supposed that the a allele is dominant); Analysis 2: AA + aa vs Aa (we supposed that the heterozygote carriers are different from the homozygote ones); Analysis 3: AA + Aa vs aa (we supposed that the A allele is dominant)). The best results are indicated in Tables 1, 2 and 3.
<p>Table 1</p>
Association studies between CCC and ASY including as covariates the gender and the polymorphism one by one
GENE
Tag SNP
Genotype groups
Association test
CCR5
rs3176763
CC vs CA + AA
p = 0.006; OR = 1.79; 95% CI: 1.18-2.70
rs3087253
AA vs AT + TT
p = 0.640; OR = 1.06; 95% CI: 0.84-1.32
rs11575815
AA + AT vs TT
p = 0.030; OR = 1.41; 95% CI: 1.03-1.92
CCL2
rs3760396
GG vs GA + AA
p = 0.373; OR = 1.13; 95% CI: 0.87-1.46
rs2857656
CC vs CG + GG
p = 0.440; OR = 1.09; 95% CI: 0.87-1.36
rs4586
TT vs TC + CC
p = 0.032; OR = 1.30; 95% CI: 1.02-1.65
rs3917891
CC vs CT + TT
p = 0.037; OR = 1.56; 95% CI: 1.03-2.37
rs2530797
AA vs AG + GG
p = 0.028; OR = 1.28; 95% CI: 1.03-1.60
rs991804
CC vs CT + TT
p = 0.493; OR = 1.17; 95% CI: 0.75-1.82
TIRAP
rs11220437
TT vs TC + CC
p = 0.155; OR = 1.21; 95% CI: 0.93-1.58
rs591163
GG + GA vs AA
p = 0.237; OR = 1.01; 95% CI: 0.79-1.30
rs8177352
AA vs AG + GG
p = 0.913; OR = 2.06; 95% CI: 0.45-9.55
rs8177375
AA vs AG + GG
p = 0.203; OR = 1.21; 95% CI: 0.90-1.61
rs8177376
AA vs AC + CC
p = 0.004; OR = 1.42; 95% CI: 1.12-1.80
rs17866704
TT vs TC + CC
p = 0.023; OR = 1.31; 95% CI: 1.04-1.66
<p>Table 2</p>
Association studies between CCC with a left ventricular ejection fraction value under 0.4% and ASY including as covariates the gender and the polymorphism one by one
GENE
Tag SNP
Genotype groups
Association test
CCR5
rs3176763
CC vs CA + AA
p = 0.005; OR = 1.88; 95% CI: 1.20-2.94
rs3087253
AA vs AT + TT
p = 0.861; OR = 1.02; 95% CI: 0.80-1.31
rs11575815
AA + AT vs TT
p = 0.138; OR = 1.29; 95% CI: 0.92-1.82
CCL2
rs3760396
GG vs GA + AA
p = 0.920; OR = 1.02; 95% CI: 0.77-1.35
rs2857656
CC vs CG + GG
p = 0.514; OR = 1.08; 95% CI: 0.85-1.39
rs4586
TT vs TC + CC
p = 0.034; OR = 1.34; 95% CI: 1.02-1.75
rs3917891
CC vs CT + TT
p = 0.053; OR = 1.55; 95% CI: 1.00-2.41
rs2530797
AA vs AG + GG
p = 0.005; OR = 1.42; 95% CI: 1.11-1.82
rs991804
CC vs CT + TT
p = 0.824; OR = 1.06; 95% CI: 0.65-1.73
TIRAP
rs11220437
TT vs TC + CC
p = 0.181; OR = 1.22; 95% CI: 0.91-1.63
rs591163
GG + GA vs AA
p = 0.188; OR = 1.39; 95% CI: 0.88-1.90
rs8177352
AA vs AG + GG
p = 0.858; OR = 1.02; 95% CI: 0.78-1.34
rs8177375
AA vs AG + GG
p = 0.174; OR = 1.25; 95% CI: 0.91-1.69
rs8177376
AA vs AC + CC
p = 0.005; OR = 1.46; 95% CI: 1.12-1.91
rs17866704
TT vs TC + CC
p = 0.087; OR = 1.25; 95% CI: 0.97-1.62
<p>Table 3</p>
Association studies performed on an independent cohort including as covariates the gender and the polymorphism one by one
CCC VS ASY
GENE
Tag SNP
Genotype groups
Association test
CCL2
rs3760396
GG vs GA + AA
p = 0.626; OR = 1.35; 95% CI: 0.40-4.55
rs2857656
CC vs CG + GG
p = 0.267; OR = 1.16; 95% CI: 0.89-1.51
rs4586
TT vs TC + CC
p = 0.128; OR = 1.25; 95% CI: 1.94-1.67
rs3917891
CC vs CT + TT
p = 0.127; OR = 1.42; 95% CI: 0.90-2.23
rs2530797
AA vs AG + GG
p = 0.007; OR = 1.46; 95% CI: 1.11-1.92
rs991804
CC vs CT + TT
p = 0.435; OR = 1.23; 95% CI: 0.73-2.09
TIRAP
rs11220437
TT vs TC + CC
p = 0.149; OR = 1.27; 95% CI: 0.92-1.75
rs591163
GG + GA vs AA
p = 0.154; OR = 1.32; 95% CI: 0.90-1.94
rs8177352
AA vs AG + GG
p = 0.278; OR = 1.20; 95% CI: 0.87-1.66
rs8177375
AA vs AG + GG
p = 0.256; OR = 1.22; 95% CI: 0.87-1.72
rs8177376
AA vs AC + CC
p = 0.037; OR = 1.36; 95% CI: 1.19-1.80
rs17866704
TT vs TC + CC
p = 0.051; OR = 1.32; 95% CI: 1.00-1.76
CCC with a left ventricular ejection fraction value under 0.4% VS ASY
GENE
Tag SNP
Genotype groups
Association test
CCL2
rs3760396
GG vs GA + AA
p = 0.392; OR = 1.84; 95% CI: 0.45-7.46
rs2857656
CC vs CG + GG
p = 0.499; OR = 1.10; 95% CI: 0.83-1.46
rs4586
TT vs TC + CC
p = 0.194; OR = 1.23; 95% CI: 0.90-1.67
rs3917891
CC vs CT + TT
p = 0.156; OR = 1.40; 95% CI: 0.88-2.24
rs2530797
AA vs AG + GG
p = 0.002; OR = 1.59; 95% CI: 1.19-2.13
rs991804
CC vs CT + TT
p = 0.876; OR = 1.05; 95% CI: 0.60-1.83
TIRAP
rs11220437
TT vs TC + CC
p = 0.265; OR = 1.21; 95% CI: 0.86-1.71
rs591163
GG + GA vs AA
p = 0.134; OR = 1.38; 95% CI: 0.91-2.10
rs8177352
AA vs AG + GG
p = 0.224; OR = 1.23; 95% CI: 0.88-1.73
rs8177375
AA vs AG + GG
p = 0.313; OR = 1.21; 95% CI: 0.84-1.74
rs8177376
AA vs AC + CC
p = 0.046; OR = 1.36; 95% CI: 1.05-1.85
rs17866704
TT vs TC + CC
p = 0.095; OR = 1.29; 95% CI: 0.96-1.74
In multivariates analyses, several polymorphisms and gender were included as covariates. All the covariates are analyzed in the same time. In a stepwise approach, the worse associated covariate (non significant) is removed and the analysis is run again up to keep only significant associated covariates.
Results and discussion
Fifteen Tag SNPs were genotyped successfully on our original cohort including ASY subjects (n = 118) and CCC patients (n = 315) (Table 4). The genotyping steps were done successfully for all the Tag SNPs. The genotype distribution of each SNP is summarized in Table 5. All the SNPs were in Hardy-Weinberg equilibrium on the ASY individuals considered as control subjects (p > 0,001) (Table 6).
<p>Table 4</p>
List of the tag SNPs genotyped on the original study population
GENE
Tag SNP
Position relative to coordinate system
Position relative to transcription start point
CCR5
rs3176763 C/A
46414281
−113
rs3087253 A/G
46418689
+4295
rs11575815 A/T
46420170
+5776
CCL2
rs3760396 G/C
32581441
−928
rs2857656 C/G
32582007
−362
rs4586 T/C
32583269
+900
rs3917891 C/T
32585687
+3318
rs2530797 A/G
32586094
+3725
rs991804 C/T
32587725
+5356
TIRAP
rs11220437 T/C
126148160
−12630
rs591163 G/A
126148432
−12358
rs8177352 A/G
126153843
−6947
rs8177375 A/G
126163064
+2274
rs8177376 A/C
126163612
+2822
rs17866704 T/C
126165757
+4967
<p>Table 5</p>
Genotype distribution on controls (ASY individuals) and cases (CCC) taking into account the gender and the left ventricular ejection fraction values
ASY
CCC
CCC (EF ≤ 0.4)
CCC (EF ≥ 0.4)
Gene
SNP
Genotype
Total
Male
Female
Total
Male
Female
Total
Male
Female
Total
Male
Female
CCR5
rs3176763
CC
110
50
59
266
167
97
169
127
42
88
34
54
(94.0%)
(96.2%)
(92.2%)
(84.4%)
(87.0%)
(80.2%)
(84.9%)
(88.2%)
(76.4%)
(89.0%)
(81.0%)
(84.4%)
CA
7
2
5
48
25
23
30
17
13
17
8
9
(6.0%)
(3.8%)
(7.8%)
(15.2%)
(13.0%)
(19.0%)
(15.1%)
(11.8%)
(23.6%)
(16.0%)
(19.0%)
(14.1%)
AA
0
0
0
1
0
1
0
0
0
1
0
1
(0.0%)
(0.0%)
(0.0%)
(0.3%)
(0.0%)
(0.8%)
(0.0%)
(0.0%)
(0.0%)
(0.9%)
(0.0%)
(1.6%)
CCR5
rs3087253
AA
46
18
28
134
81
52
80
61
19
52
20
32
(41.4%)
(36.7%)
(45.9%)
(43.9%)
(43.5%)
(44.4%)
(41.0%)
(43.3%)
(35.2%)
(51.0%)
(50.0%)
(51.6%)
AG
47
22
24
119
73
46
80
52
28
34
16
18
(42.3%)
(44.9%)
(39.3%)
(39.0%)
(39.2%)
(39.3%)
(41.0%)
(36.9%)
(51.9%)
(33.3%)
(40.0%)
(29.0%)
GG
18
9
9
52
32
19
35
28
7
16
4
12
(16.2%)
(18.4%)
(14.8%)
(17.0%)
(17.2%)
(16.2%)
(17.9%)
(19.9%)
(13.0%)
(15.7%)
(10.0%)
(19.4%)
CCR5
rs11575815
AA
51
25
25
158
97
60
104
74
22
51
22
29
(45.1%)
(49.0%)
(41.0%)
(51.3%)
(51.6%)
(50.8%)
(52.8%)
(52.1%)
(53.7%)
(50.0%)
(53.7%)
(47.5%)
AT
42
20
22
120
70
49
71
50
16
43
16
27
(37.2%)
(39.2%)
(36.1%)
(39.0%)
(37.2%)
(41.5%)
(36.0%)
(35.2%)
(39.0%)
(42.2%)
(39.0%)
(44.3%)
TT
20
6
14
30
21
9
22
18
3
8
3
5
(17.7%)
(11.8%)
(23.0%)
(9.7%)
(11.2%)
(7.6%)
(11.2%)
(12.7%)
(7.3%)
(7.8%)
(7.3%)
(8.2%)
CCL2
rs3760396
GG
87
41
45
247
152
94
153
112
41
87
36
51
(75.7%)
(80.4%)
(71.4%)
(79.9%)
(80.9%)
(79.0%)
(77.7%)
(78.9%)
(74.5%)
(85.3%)
(90.0%)
(82.3%)
GC
27
9
18
60
35
24
43
29
14
14
4
10
(23.5%)
(17.6%)
(28.6%)
(19.4%)
(18.6%)
(20.2%)
(21.8%)
(20.4%)
(25.5%)
(13.7%)
(10.0%)
(16.1%)
CC
1
1
0
2
1
1
1
1
0
1
0
1
(0.9%)
(2.0%)
(0.0%)
(0.6%)
(0.5%)
(0.8%)
(0.5%)
(0.7%)
(0.0%)
(1.0%)
(0.0%)
(1.6%)
CCL2
rs2857656
CC
50
24
25
122
70
50
77
52
25
42
17
25
(44.2%)
(48.0%)
(40.3%)
(39.7%)
(37.6%)
(42.0%)
(39.3%)
(36.9%)
(45.5%)
(41.2%)
(42.5%)
(40.3%)
CG
51
22
29
150
92
58
96
72
24
51
18
33
(45.1%)
(44.0%)
(46.8%)
(48.9%)
(49.5%)
(48.7%)
(49.0%)
(51.1%)
(43.6%)
(50.0%)
(45.0%)
(53.2%)
GG
12
4
8
35
24
11
23
17
6
9
5
4
(10.6%)
(8.0%)
(12.9%)
(11.4%)
(12.9%)
(9.2%)
(11.7%)
(12.1%)
(10.9%)
(8.8%)
(12.5%)
(6.5%)
CCL2
rs4586
TT
38
20
18
74
41
32
46
31
15
26
9
17
(34.5%)
(40.8%)
(30.0%)
(24.0%)
(21.7%)
(27.4%)
(23.4%)
(21.8%)
(27.3%)
(25.5%)
(22.0%)
(27.9%)
TC
53
23
29
148
90
57
94
70
24
52
19
33
(48.2%)
(49.9%)
(48.3%)
(48.1%)
(47.6%)
(48.7%)
(47.7%)
(49.3%)
(43.6%)
(51.0%)
(46.3%)
(54.1%)
CC
19
6
13
86
58
28
57
41
16
26
13
11
(17.3%)
(12.2%)
(21.7%)
(27.9%)
(30.7%)
(23.9%)
(28.9%)
(28.9%)
(29.1%)
(25.5%)
(31.7%)
(18.0%)
CCL2
rs3917891
CC
107
47
59
264
161
101
166
120
46
91
37
54
(93.9%)
(90.4%)
(96.7%)
(86.0%)
(86.1%)
(85.6%)
(85.6%)
(85.7%)
(85.2%)
(88.3%)
(90.2%)
(87.1%)
CT
7
5
2
41
25
16
26
19
7
12
4
8
(6.1%)
(9.6%)
(3.3%)
(13.4%)
(13.4%)
(13.6%)
(13.4%)
(13.6%)
(13.0%)
(11.7%)
(9.9%)
(12.9%)
TT
0
0
0
2
1
1
2
1
1
0
0
0
(0.0%)
(0.0%)
(0.0%)
(0.7%)
(0.5%)
(0.8%)
(1.0%)
(0.7%)
(1.9%)
(0.0%)
(0.0%)
(0.0%)
CCL2
rs2530797
AA
47
13
33
163
104
58
110
81
29
47
19
28
(41.6%)
(25.5%)
(54.1%)
(52.9%)
(55.6%)
(48.7%)
(55.8%)
(57.0%)
(52.7%)
(45.6%)
(47.5%)
(44.4%)
AG
52
33
19
115
68
46
69
51
18
44
16
28
(46.0%)
(64.7%)
(31.1%)
(37.3%)
(36.4%)
(38.7%)
(35%)
(35.9%)
(32.7%)
(42.7%)
(40%)
(44.4%)
GG
14
5
9
30
15
15
18
10
8
12
5
7
(12.4%)
(9.8%)
(14.8%)
(9.7%)
(8.0%)
(12.6%)
(9.1%)
(7.0%)
(14.5%)
(11.7%)
(12.5%)
(11.1%)
CCL2
rs991804
CC
51
25
25
120
67
51
78
52
26
40
15
25
(45.5%)
(48.1%
(42.4%)
(41.2%)
(38.3%)
(44.7%)
(42.6%)
(39.4%)
(51.0%)
(40.4%)
(39.5%)
(41.0%)
CT
53
24
29
148
92
56
88
67
21
54
20
34
(47.3%)
(46.2%)
(49.2%)
(50.9%)
(52.6%)
(51.1%)
(48.1%)
(50.8%)
(41.2%)
(54.5%)
(52.6%)
(55.7%)
TT
8
3
5
23
16
7
17
13
4
5
3
2
(7.1%)
(5.8%)
(8.5%)
(7.9%)
(9.1%)
(6.1%)
(9.3%)
(9.8%)
(7.8%)
(5.1%)
(7.9%)
(3.3%)
TIRAP
rs11220437
TT
92
44
47
229
142
85
146
106
40
74
31
43
(80.0%)
(84.6%)
(75.8%)
(73.9%)
(74.7%)
(72.0%)
(73.7%)
(73.6%)
(74.1%)
(71.8%)
(75.6%)
(69.4%)
TC
22
8
14
76
45
31
48
36
12
28
9
19
(19.1%)
(15.4%)
(22.6%)
(24.5%)
(23.7%)
(26.3%)
(24.2%)
(25.0%)
(22.2%)
(27.2%)
(22.0%)
(30.6%)
CC
1
0
1
5
3
2
4
2
2
1
1
0
(9.0%)
(0.0%)
(1.6%)
(1.6%)
(1.6%)
(1.7%)
(2.0%)
(1.4%)
(3.7%)
(1.0%)
(2.4%)
(0.0%)
TIRAP
rs591163
GG
51
23
28
158
95
82
104
74
20
52
30
32
(46.4%)
(46.9%)
(46.7%)
(51.5%)
(50.5%)
(53.0%)
(53.1%)
(52.1%)
(50.0%)
(51.5%)
(55.6%)
(52.5%)
GA
44
19
24
120
76
43
75
56
16
38
19
22
(40.0%)
(38.8%)
(40.0%)
(39.1%)
(40.4%)
(36.8%)
(38.3%)
(39.4%)
(40.0%)
(37.6%)
(35.2%)
(36.1%)
AA
15
7
8
29
17
12
17
12
4
11
5
7
(13.6%)
(14.3%)
(13.3%)
(9.4%)
(9.0%)
(10.3%)
(8.7%)
(8.5%)
(10.0%)
(10.9%)
(9.3%)
(11.5%)
TIRAP
rs8177352
AA
83
38
44
225
132
91
140
98
42
80
31
49
(73.5%)
(4.5%)
(72.1%)
(73.3%)
(70.6%)
(77.1%)
(71.4%)
(69.5%)
(76.4%)
(77.7%)
(75.6%)
(79.0%)
AG
28
12
16
73
48
25
47
36
11
23
10
13
(24.8%)
(23.5%)
(26.2%)
(23.8%)
(25.7%)
(21.2%)
(24.0%)
(25.5%)
(20.0%)
(22.3%)
(24.4%)
(21.0%)
GG
2
60
1
9
7
2
9
7
2
0
0
0
(1.8%)
(23.5%)
(1.6%)
(2.9%)
(3.7%)
(1.7%)
(4.6%)
(5.0%)
(3.6%)
(0.0%)
(0.0%)
(0.0%)
TIRAP
rs8177375
AA
95
45
49
246
153
91
156
115
41
82
34
48
(84.1%)
(88.2%)
(80.3%)
(79.4%)
(81.4%)
(75.8%)
(79.2%)
(81.0%)
(74.5%)
(78.8%)
(82.9%)
(76.2%)
AG
16
5
11
60
33
27
39
26
13
20
6
14
(14.2%)
(9.8%)
(18.0%)
(19.4%)
(17.6%)
(22.5%)
(19.8%)
(18.3%)
(23.6%)
(19.2%)
(14.6%)
(22.2%)
GG
2
1
1
4
2
2
2
1
1
2
1
1
(1.8%)
(2.0%)
(1.6%)
(1.2%)
(1.0%)
(1.7%)
(1.0%)
(0.7%)
(1.8%)
(1.9%)
(2.4%)
(1.6%)
TIRAP
rs8177376
AA
63
26
37
230
143
85
149
110
39
74
29
45
(54.9%)
(57.8%)
(60.7%)
(75.4%)
(77.7%)
(71.4%)
(76.8%)
(79.1%)
(70.9%)
(71.8%)
(72.5%)
(71.4%)
AC
40
18
22
70
40
30
44
28
16
25
11
14
(37.7%)
(40.0%)
(36.1%)
(23.0%)
(21.7%)
(25.2%)
(22.7%)
(20.1%)
(29.1%)
(24.3%)
(27.5%)
(22.2%)
CC
3
1
2
5
1
4
1
1
0
4
0
4
(2.8%)
(2.2%)
(3.3%)
(1.6%)
(0.5%)
(3.4%)
(0.5%)
(0.7%)
(0.0%)
(3.9%)
(0.0%)
(6.3%)
TIRAP
rs17866704
TT
80
35
44
175
103
71
115
80
35
54
20
34
(70.8%)
(70.0%)
(71.0%)
(57.4%)
(55.4%)
(60.7%)
(59.0%)
(56.7%)
(64.8%)
(53.5%)
(50.0%)
(55.7%)
TC
32
15
17
106
64
41
63
46
17
40
16
24
(28.3%)
(30.0%)
(27.4%)
(34.8%)
(34.4%)
(35.0%)
(32.3%)
(32.6%)
(31.5%)
(39.6%)
(40%)
(39.3%)
CC
1
0
1
24
19
5
17
15
2
7
4
3
(0.9%)
(0.0%)
(1.6%)
(7.9%)
(10.2%)
(4.3%)
(8.7%)
(10.6%)
(3.7%)
(6.9%)
(10.0%)
(4.9%)
<p>Table 6</p>
Hardy-Weinberg equilibrium test
GENE
Tag SNP
Chi2
p
CCR5
rs3176763 C/A
0.111257738
0.9458
rs3087253 A/G
1.014584489
0.6021
rs11575815 A/T
0.111257738
0.9458
CCL2
rs3760396 G/C
0.491314613
0.7821
rs2857656 C/G
0.035595421
0.9823
rs4586 T/C
0.004981781
0.9975
rs3917891 C/T
0.114371123
0.9444
rs2530797 A/G
0.004293958
0.9978
rs991804 C/T
1.35646743
0.5075
TIRAP
rs11220437 T/C
0.063307752
0.9688
rs591163 G/A
1.190573349
0.5514
rs8177352 A/G
0.042221875
0.9791
rs8177375 A/G
1.69100575
0.4293
rs8177376 A/C
1.294967649
0.5233
rs17866704 T/C
1.314206075
0.5183
Polymorphisms rs3176763C/A and rs11575815A/T, around the CCR5 gene, are associated to an increased risk of CCC
Three tag SNPs were genotyped for the CCR5 gene. In the CCC subjects group, 266 (84.4%) subjects carried the rs3176763C/C genotype whereas 110 (94.0%) of the ASY controls carried this genotype. This difference was significant in an univariate analysis including also the gender as covariate (p = 0.006; OR = 1.79; 95% CI: 1.18-2.70) (see Table 1).
For the rs11575815A/T polymorphism, 278 (90.3%) CCC subjects carried the genotypes rs11575815A/A or rs11575815A/T versus 93 (82.3%) for the ASY controls. This difference was significant (p = 0.030; OR = 1.41; 95% CI: 1.03-1.92) (see Table 1).
We performed a multivariate analysis (binary regression, stepwise procedure) to confirm the associations found previously in univariate analysis. Similarly to the univariate analysis, the genotypes were considered as binary variables. In this analysis, we included rs3176763C/A, rs11575815A/T and the gender as covariates. Polymorphism rs3176763C/A (p = 0.014; OR = 1.69; 95% CI: 1.11-2.57) and the gender (p = 0.002; OR = 2.04; 95% CI: 1.31-3.19) were still significantly associated to CCC (see Table 7). A trend of association was detected for rs11575815A/T (p = 0.077; OR = 1.33; 95% CI: 0.97-1.82).
<p>Table 7</p>
Multivariate stepwise binary logistic regression analysis between CCC and ASY including as covariates the gender and the polymorphisms associated in univariate analysis gene by gene
GENE:
CCR5
Step
Covariates
Groups
Association test
Step1
gender
Male vs Female
p = 0.002; OR = 2.042; 95% CI: 1.31-3.19
rs3176763
CC vs CA + AA
p = 0.014; OR = 1.689; 95% CI: 1.11-2.57
rs11575815
AA + AT vs TT
p = 0.077; OR = 1.328; 95% CI: 1.03-1.82
Step2
gender
Male vs Female
p = 0.001; OR = 2.058; 95% CI: 1.32-3.21
rs3176763
CC vs CA + AA
p = 0.007; OR = 1.766; 95% CI: 1.16-2.68
rs11575815
AA + AT vs TT
Excluded
GENE:
CCL2
Step
Covariates
Groups
Association test
Step1
gender
Male vs Female
p = 0.002; OR = 2.056; 95% CI: 1.31-3.23
rs2530797
AA vs AG + GG
p = 0.162; OR = 1.198; 95% CI: 1.07-1.54
rs4586
TT vs TC + CC
p = 0.348; OR = 1.138; 95% CI: 1.15-1.49
rs3917891
CC vs CT + TT
p = 0.131; OR = 1.392; 95% CI: 1.1-2.140
Step2
gender
Male vs Female
p = 0.002; OR = 2.070; 95% CI: 1.32-3.25
rs2530797
AA vs AG + GG
p = 0.051; OR = 1.258; 95% CI: 1.00-1.59
rs3917891
CC vs CT + TT
p = 0.095; OR = 1.435; 95% CI: 1.06-2.19
rs4586
TT vs TC + CC
Excluded
Step3
gender
Male vs Female
p = 0.001; OR = 2.091; 95% CI: 1.33-3.28
rs2530797
AA vs AG + GG
p = 0.022; OR = 1.303; 95% CI: 1.04-1.64
rs4586
TT vs TC + CC
Excluded
rs3917891
CC vs CT + TT
Excluded
GENE:
TIRAP
Step
Covariates
Groups
Association test
Step1
gender
Male vs Female
p = 0.002; OR = 2.062; 95% CI: 1.30-3.27
rs8177376
AA vs AC + CC
p = 0.013; OR = 1.357; 95% CI: 1.06-1.73
rs17866704
TT vs TC + CC
p = 0.039; OR = 1.298; 95% CI: 1.01-1.66
When we compared the ASY subjects to severe CCC patients (left ventricular ejection fraction value under 0.4%), only the association of rs3176763C/A was maintained in univariate analysis (p = 0.005; OR = 1.88; 95% CI: 1.20-2.94) (see Table 2). These two markers (rs3176763C/A and rs11575815A/T) did not discriminate moderate CCC from severe CCC (p > 0.5).
Polymorphisms rs4586T/C, rs3917891C/T and rs2530797A/G, around the CCL2 gene, are associated to an increased risk of CCC
Six tag SNPs were genotyped for the CCL2 gene. In the CCC subjects group, 74 (24.0%) carried the rs4586T/T genotype whereas 38 (34.5%) of the ASY controls carried this genotype. This difference was significant in an univariate analysis (p = 0.032; OR = 1.30; 95% CI: 1.02-1.65) (see Table 1).
For the rs3917891C/T polymorphism, 264 (86.0%) CCC subjects carried the rs3917891C/C genotype versus 107 (93.9%) for the ASY controls. This difference was significant (p = 0.037; OR = 1.56; 95% CI: 1.03-2.37) (see Table 1).
For the rs2530797A/G polymorphism, 163 (52.9%) CCC subjects carried the rs2530797A/A genotype versus 47 (41.6%) for the ASY controls. This difference was significant (p = 0.028; OR = 1.28; 95% CI: 1.03-1.60) (see Table 1).
The same polymorphisms were associated when we compared the ASY subjects to severe CCC patients (rs4586T/C: p = 0.034; OR = 1.34; 95% CI: 1.02-1.75; rs3917891C/T: p = 0.053; OR = 1.55; 95% CI: 1.00-2.41; rs2530797A/G: p = 0.005; OR = 1.42; 95% CI: 1.11-1.82) (see Table 2).
We performed multivariate analysis including these three polymorphisms and the gender as covariates. When we compared the ASY subjects to CCC patients, only the polymorphism rs2530797A/G and the gender remained significantly associated (rs2530797A/G: p = 0.022; OR = 1.30; 95% CI: 1.04-1.64; gender: p = 0.001; OR = 2.09; 95% CI: 1.33-3.28) (see Table 7).
The same result was obtained, when we compared the ASY subjects to severe CCC patients (rs2530797A/G: p = 8.51×10-7; OR = 1.46; 95% CI: 1.13-1.88; gender: p = 0.004; OR = 3.59; 95% CI: 2.16-5.97). These three markers (rs4586T/C, rs3917891C/T and rs2530797A/G) did not discriminate moderate CCC from severe CCC (p > 0.16).
Polymorphism rs8177376A/C, around the MAL/TIRAP gene, is associated to an an increased risk of CCC
Six tag SNPs were genotyped for the MALTIRAP gene. For the rs8177376A/C polymorphism, 230 (75.4%) CCC subjects carried the rs8177376A/A genotype versus 63 (54.9%) for the ASY controls. This difference was significant (p = 0.004; OR = 1.42; 95% CI: 1.12-1.80) (see Table 1). The same result was obtained when the analysis was restricted to severe CCC (p = 0.005; OR = 1.46; 95% CI: 1.12-1.91) (see Table 2).
A statistically significant difference was also detected for the rs17866704T/C polymorphism (p = 0.023; OR = 1.31; 95% CI: 1.04-1.66) (see Table 1). In our cohort, 175 ((57.4%) CCC subjects carried the rs17866704T/T genotype versus 80 (70.8%) for the ASY controls. The two SNPs remained associated in a multivariate analysis (see Table 7).
Some trend of association was detected for the rs17866704T/C polymorphism when we compared the ASY subjects to the severe CCC patients (p = 0.087; OR = 1.25; 95% CI: 0.97-1.62) (see Table 2). The rs8177376A/C marker did not discriminate moderate CCC from severe CCC (p > 0.57).
The associations of the CCL2 and MAL/TIRAP genes were confirmed in a cohort from the original reports
The original data reporting association between the CCL2 and TIRAP genes were done by Ramasawmy et al.
41
42
. These studies were done on 169 patients with CCC and 76 T. cruzi infected ASY individuals. Our present study population is partially overlapping with the original one described by Ramasawmy et al. So, we repeated the analysis for these two genes on our cohort after removing the common subjects. This independent cohort includes 110/118 ASY subjects and 281/315 CCC patients. Of 281 patients with CCC, 192 had severe ventricular dysfunction and were classified as having severe CCC. The genotype distribution of the CCL2 and TIRAP Tag SNPs, on this independent cohort, is summarized in Table 8. In association studies, the gender was also included as covariates.
<p>Table 8</p>
Genotype distribution on our independent cohort which included 110 ASY controls and 281 cases (CCC) taking into account the gender and the left ventricular ejection fraction values
ASY
CCC
CCC (EF ≤ 0.4)
CCC (EF ≥ 0.4)
Gene
SNP
Genotype
Total
Male
Female
Total
Male
Female
Total
Male
Female
Total
Male
Female
CCL2
rs3760396
GG
61
30
30
208
128
79
140
99
41
76
28
36
(81.3%)
(81.1%)
(81.1%)
(80.0%)
(80.9%)
(76.7%)
(78.7%)
(80.5%)
(74.5%)
(86.4%)
(93.3%)
(78.3%)
GC
13
6
7
50
26
23
37
23
14
11
2
9
(17.3%)
(16.2%)
(18.9%)
(19.4%)
(19.2%)
(22.3%)
(20.8%)
(18.7%)
(25.5%)
(12.5%)
(6.7%)
(19.6%)
CC
1
1
0
2
1
1
1
1
0
1
0
1
(1.4%)
(2.7%)
(0.0%)
(0.6%)
(0.9%)
(1.0%)
(0.5%)
(0.8%)
(0.0%)
(1.1%)
(0.0%)
(2.1%)
CCL2
rs2857656
CC
34
16
17
101
57
42
71
46
25
28
11
17
(46.6%)
(44,4%)
(47,2%)
(38.7%)
(36.8%)
(42.0%)
(39.9%)
(37,4%)
(45.5%)
(36,4%)
(36,7%)
(36,2%)
CG
36
20
16
133
59
54
86
62
24
45
16
29
(49.3%)
(55,6%)
(44,4%)
(51%)
(51%)
(51,9%)
(48,3%)
(50,4%)
(43.6%)
(58,4%)
(53,3%)
(61,7%)
GG
3
0
3
27
19
8
21
15
6
4
3
1
(4,1%)
(0%)
(8,3%)
(10.3%)
(12.3%)
(7,7%)
(11.8%)
(12.2%)
(10.9%)
(5,2%)
(10%)
(2,1%)
CCL2
rs4586
TT
23
13
10
62
34
27
44
29
15
17
5
12
(32,4%)
(37,1%)
(28,6%)
(23,8%)
(21.8%)
(26,5%)
(24,6%)
(23,4%)
(27.3%)
(22,4%)
(16,7%)
(26,1%)
TC
39
20
18
130
78
51
84
60
24
44
17
27
(54,9%)
(57,1%)
(51,4%)
(50%)
(50%)
(50%)
(46,9%)
(48,4%)
(43.6%)
(57,9%)
(56,7%)
(58,7%)
CC
9
2
7
68
44
24
51
35
16
15
8
7
(12,7%)
(5,7%)
(20%)
(22.2%)
(28,2%)
(23.5%)
(28.5%)
(28.2%)
(29.1%)
(19,7%)
(26,7%)
(15,2%)
CCL2
rs3917891
CC
69
34
34
220
132
86
150
104
46
66
27
39
(92%)
(89,5%)
(94,4%)
(84.9%)
(85,2%)
(84,3%)
(84,7%)
(84,6%)
(85.2%)
(86,8%)
(90%)
(84,8%)
CT
6
4
2
37
22
15
25
18
7
10
3
7
(8%)
(10,5%)
(5,6%)
(14,3%)
(14,2%)
(14,7%)
(14,1%)
(14.6%)
(13.0%)
(13,2%)
(10%)
(15,2%)
TT
0
0
0
2
1
1
2
1
1
0
0
0
(0%)
(0%)
(0.0%)
(0.8%)
(0.6%)
(1%)
(1.1%)
(0.8%)
(1.9%)
(0.0%)
(0.0%)
(0.0%)
CCL2
rs2530797
AA
25
7
17
132
81
50
96
67
29
32
12
20
(33.8%)
(18.9%)
(47.2%)
(50.6%)
(51.9%)
(48.5%)
(53.6%)
(54.0%)
(52,7%)
(41.6%)
(40.0%)
(42.6%)
AG
40
26
14
104
62
41
65
47
18
38
15
23
(54.1%)
(70.3%)
(38.9%)
(39.8%)
(39.7%)
(39.8%)
(36.3%)
(37.9%)
(32.7%)
(49.4%)
(50%)
(48.9%)
GG
9
4
5
25
13
12
18
10
8
7
3
4
(12.2%)
(10.8%)
(13.9%)
(9.6%)
(8.3%)
(11.7%)
(10.1%)
(8.1%)
(14,5%)
(9.1%)
(10.0%)
(8.5%)
CCL2
rs991804
CC
35
16
18
99
55
42
71
45
26
26
10
16
(47.6%)
(42,1%)
(50%)
(40,9%)
(38.7%)
(42,9%)
(43%)
(39.5%)
(51.0%)
(36,1%)
(37%)
(35,6%)
CT
36
21
15
122
72
50
77
56
21
43
15
28
(48%)
(55,3%)
(41,7%)
(50.4%)
(50,7%)
(51%)
(46,7%)
(49,1%)
(41.2%)
(59,7%)
(55.6%)
(62,2%)
TT
4
1
3
21
15
6
17
13
4
3
2
1
(5,3%)
(2,6%)
(8.3%)
(8,7%)
(10,6%)
(6.1%)
(10.3%)
(11,4%)
(7.8%)
(4,2%)
(7.4%)
(2,2%)
TIRAP
rs11220437
TT
61
30
30
189
114
73
132
92
40
51
20
31
(81,3%)
(79,8%)
(83,3%)
(73.0%)
(73,5%)
(71,6%)
(74,2%)
(74,2%)
(74.1%)
(68%)
(69%)
(67,4%)
TC
13
8
5
65
38
27
42
30
12
23
8
15
(17,3%)
(21,1%)
(13,9%)
(25,1%)
(24,5%)
(26.5%)
(23,6%)
(24,2%)
(22.2%)
(30,7%)
(27,6%)
(32,6%)
CC
1
0
1
5
3
2
4
2
2
1
1
0
(1,3%)
(0.0%)
(2,8%)
(1.9%)
(1.9%)
(2%)
(2.2%)
(1.6%)
(3.7%)
(1.3%)
(3.4%)
(0.0%)
TIRAP
rs591163
GG
27
14
13
132
77
54
91
61
30
40
16
24
(38%)
(40%)
(37,1%)
(50,8%)
(49,7%)
(52,4%)
(51,4%)
(49,6%)
(55,6%)
(51.9%)
(53,3%)
(51,1%)
GA
33
15
17
103
63
39
70
51
19
29
11
18
(46,5%)
(42,9%)
(48,6%)
(39.6%)
(40,6%)
(37,9%)
(39,5%)
(41,5%)
(35,2%)
(37.7%)
(36,7%)
(38,3%)
AA
11
6
5
25
15
10
16
11
5
8
3
5
(15,5%)
(17,1%)
(14,3%)
(9.6%)
(9.7%)
(9,7%)
(9%)
(8.9%)
(9,3%)
(10.4%)
(10%)
(10,6%)
TIRAP
rs8177352
AA
59
30
28
192
111
79
129
87
42
59
22
37
(80,8%)
(81,1%)
(80%)
(74,1%)
(71.6%)
(77.5%)
(72,5%)
(70,7%)
(76.4%)
(77.6%)
(73,3%)
(80,4%)
AG
13
6
7
58
37
21
40
29
11
17
8
9
(17.8%)
(16,2%)
(20%)
(22,4%)
(23,9%)
(20,6%)
(22,5%)
(23,6%)
(20.0%)
(22.4%)
(26,7%)
(19,6%)
GG
1
1
0
9
7
2
9
7
2
0
0
0
(1.4%)
(2,7%)
(0%)
(3,5%)
(4,5%)
(2%)
(5,1%)
(5.7%)
(3.6%)
(0.0%)
(0.0%)
(0.0%)
TIRAP
rs8177375
AA
61
32
28
203
124
77
140
99
41
58
24
34
(83,6%)
(86,5%)
(80%)
(77,8%)
(80%)
(74%)
(78,7%)
(80,5%)
(74.5%)
(75,3%)
(80%)
(72,3%)
AG
10
4
6
54
29
25
36
23
13
17
5
12
(13,7%)
(10,8%)
(17,1%)
(20,7%)
(18,7%)
(24%)
(20,2%)
(18.7%)
(23.6%)
(22,1%)
(16,7%)
(25,5%)
GG
2
1
1
4
2
2
2
1
1
2
1
1
(2,7%)
(2,7%)
(2,9%)
(1.5%)
(1.3%)
(1.9%)
(1.1%)
(0.8%)
(1.8%)
(2,6%)
(3,3%)
(2,1%)
TIRAP
rs8177376
AA
42
19
23
195
121
72
134
95
39
56
24
32
(61,8%)
(59,4%)
(63,9%)
(75,6%)
(78,6%)
(70,6%)
(75,7%)
(77,9%)
(70.9%)
(73,3%)
(80%)
(69,6%)
AC
25
13
12
60
32
28
42
26
16
18
6
12
(36,8%)
(40.6%)
(33,3%)
(23.3%)
(20,8%)
(27,5%)
(23,7%)
(21,3%)
(29.1%)
(23,7%)
(20%)
(26,1%)
CC
1
0
1
3
1
2
1
1
0
2
0
2
(1,5%)
(0%)
(2,8%)
(1.2%)
(0.6%)
(2%)
(0.6%)
(0.8%)
(0.0%)
(2,6%)
(0.0%)
(4.3%)
TIRAP
rs17866704
TT
54
27
26
150
85
71
105
70
35
41
14
27
(72%)
(75%)
(68,4%)
(58,4%)
(55,2%)
(60.7%)
(59,7%)
(57,4%)
(64.8%)
(54,7%)
(46,7%)
(60%)
TC
20
9
11
88
55
41
59
42
17
27
12
15
(26,7%)
(25%)
(28,9%)
(34,2%)
(35,7%)
(35.0%)
(33,5%)
(34,4%)
(31.5%)
(36%)
(40%)
(33,3%)
CC
1
0
1
19
14
5
12
10
2
7
4
3
(1,3%)
(0.0%)
(2.6%)
(7,4%)
(9,1%)
(4.3%)
(6,8%)
(8,2%)
(3.7%)
(9,3%)
(13,3%)
(6,7%)
For the CCL2rs2530797A/G polymorphism, 132 (50.6%) CCC subjects carried the rs2530797A/A genotype versus 25 (33.8%) for the ASY controls (see Table 8). This difference was significant (p = 0.007; OR = 1.4 p = 0.007; OR = 1.46; 95% CI: 1.11-1.926) (see Table 8). The same polymorphism remained associated when we compared the ASY subjects to severe CCC patients (p = 0.002; OR = 1.59; 95% CI: 1.19-2.13) (see Table 3).
For the MAL/TIRAPrs8177376A/C polymorphism, 195 (75.6%) CCC subjects carried the rs8177376A/A genotype versus 42 (61.8%) for the ASY controls (see Table 8). This difference was significant on the whole independent cohort (p = 0.037; OR = 1.36; 95% CI: 1.19-1.80) (see Table 3). The same result was obtained when the analysis was restricted to severe CCC (p = 0.046; OR = 1.36; 95% CI: 1.05-1.85) (see Table 3).
A trend of association was detected for the rs17866704T/C polymorphism in both analyses (p = 0.051; OR = 1.32; 95% CI: 1.00-1.76) (see Table 3) and (p = 0.095; OR = 1.29; 95% CI: 0.96-1.74) (see Table 3).
In order to detect interaction between the candidate genes a multivariate stepwise binary logistic regression analysis was performed on ASY subjects and CCC patients (see Table 9). In this analysis, we included the gender, rs11575815A/T, rs2530797A/G, rs8177376A/C and rs17866704T/C as covariates. Polymorphisms CCR5rs3176763C/A (p = 0.007; OR = 1.879; 95% CI: 1.19-1.89), TIRAP rs8177376A/C (p = 0.007; OR = 1.393; 95% CI: 1.09-1.77) and the gender (p = 0.001; OR = 2.226; 95% CI: 1.39-3.55) were still significantly associated to CCC (see Table 9). However, if we want to add a significant number of genes and polymorphisms at the first step of the multivariate analysis, the study population (which is one of the largest described so far) is underpowered. So, we are working toward obtaining a cohort between 1,500 and 2,000 subjects that would enable us to assess whether possessing a given combination of alleles in several SNPs contribute more strongly for prognosis than the individual SNPs.
<p>Table 9</p>
Multivariate stepwise binary logistic regression analysis between CCC and ASY including as covariates the gender and the polymorphisms associated in all the previous multivariate analysis
Step
Covariates
Groups
Association test
Step1
Gender
Male vs Female
p = 0.001; OR = 2.179; 95% CI: 1.36-3.49
CCR5rs3176763
CC vs CA + AA
p = 0.014; OR = 1.763; 95% CI: 1.12-2.77
TIRAP rs8177376
AA vs AC + CC
p = 0.014; OR = 1.363; 95% CI: 1.07-1.74
TIRAP rs17866704
TT vs TC + CC
p = 0.048; OR = 1.291; 95% CI: 1.01-1.66
CCL2rs2530797
AA vs AG + GG
p = 0.114; OR = 1.212; 95% CI: 1.04-1.54
TIRAP rs8177376
AA vs AC + CC
p = 0.014; OR = 1.363; 95% CI: 1.07-1.74
Step2
Gender
Male vs Female
p = 0.001; OR = 2.179; 95% CI: 1.36-3.48
CCR5rs3176763
CC vs CA + AA
p = 0.008; OR = 1.842; 95% CI: 1.17-2.88
TIRAP rs8177376
AA vs AC + CC
p = 0.015; OR = 1.356; 95% CI: 1.06-1.73
TIRAP rs17866704
TT vs TC + CC
p = 0.064; OR = 1.267; 95% CI: 1.01-1.63
CCL2rs2530797
AA vs AG + GG
Excluded
Step3
Gender
Male vs Female
p = 0.001; OR = 2.226; 95% CI: 1.39-3.55
CCR5rs3176763
CC vs CA + AA
p = 0.007; OR = 1.879; 95% CI: 1.19-1.89
TIRAP rs8177376
AA vs AC + CC
p = 0.007; OR = 1.393; 95% CI: 1.09-1.77
TIRAP rs17866704
TT vs TC + CC
Excluded
CCL2rs2530797
AA vs AG + GG
Excluded
We conducted an association study on several previously studied candidate genes on a Brazilian population. Whereas previously studies were done on a limited number of subjects (CCC patients ranges from 27 to 169, ASY controls ranges from 27 to 132) our study was done on a main cohort including 433 Chagas disease patients from the states of Sao Paulo, Minas Gerais and Bahia states. These patients were classified as seropositive ASY (n = 118) or as having CCC (n = 315). Whereas, previous studies were done on a limited number of SNPs, here, a Tag SNPs approach was applied to catch all the genetic information from each candidate gene.
For the CCR5 gene, two markers were associated to CCC (rs3176763C/A and rs11575815A/T). The association of rs3176763C/A was confirmed in a multivariate analysis or in a univariate analysis focusing only on severe CCC cases. rs3176763C/A polymorphism is located in the promoter of the gene and may affect the binding of transcription factors. Although these SNPs were not studied before, is in line with the literature in studies performed in other Latin American countries with diverse ethnic compositions, where several SNPs were located in the 5′UTR of the CCR5 gene where they may influence binding of regulatory elements to gene expression control regions
22
47
48
61
. As suggested by Florez et al., these polymorphisms do not act independently
61
. Multiple polymorphic changes in the promoter may influence in a differential way the levels of CCR5 expression and the type of cell in which it is expressed. So, it’s more appropriate to talk about a susceptibility haplotype rather than individual SNPs. The content and the length of this haplotype may vary from one population to the other. The subsets of patients that develop Chagas cardiomyopathy display an exacerbated Th1 immune response. The relevance of the CCR5 and CXCR3 chemokine–chemokine receptor axis in Th1 cell migration to the heart has been demonstrated in experimental models
62
63
64
and in CCC
22
.
For the CCL2 gene, three markers were associated with CCC (rs4586T/C, rs3917891C/T and rs2530797A/G). Only the rs2530797A/G polymorphism remains associated into multivariate analysis. The rs4586C/T polymorphism is a synonymous marker, whereas the two other SNPs are located into the 3′ region of the gene and may affect stability of the transcript or the binding of regulatory elements. The previous associated marker reported by Ramasawmy et al. is located into the promoter region (CCL2-2518A-G known as rs1024611)
42
. These results are absolutely not in discrepancy. Indeed, our tag SNPs were selected on the CEU and YRI reference populations. In these two reference populations the rs2530797A/G and rs1024611 are in strong linkage disequilibrium (previous associated marker) (D’ = 1). So the genetic involvement of the CCL2 gene in the control of the human susceptibility to chronic disease is confirmed. Patients with severe Chagas disease had elevated plasma concentrations of TNF-α and CCL2. Moreover, there is a good correlation between levels of these proteins (especially TNF-α) and the degree of left ventricular dysfunction
14
. Real-time quantitative PCR analysis in human CCC myocardium showed that the gene expression levels of CCL2 was selectively upregulated
12
, reinforcing the importance of regulation of CCL2 expression in the pathogenesis of CCC.
For the TIRAP gene, only one marker, located into the 3′UTR region of the gene, was strongly associated (rs8177376A/C) and may affect stability of the transcript or the binding of regulatory elements. This result is in line with previous association reported by Ramasawmy et al.
41
who reported a non-synonimous polymorphism at a coding region (TIRAP975C/T, S180L known also as rs8177374). Indeed these two SNPs (rs8177376 and rs8177374) are in strong linkage disequilibrium. This gene encodes for a TIR adaptor protein involved in the TLR4 signaling pathway of the immune system. It activates NF-kappa-B, MAPK1, MAPK3 and JNK, which promote cytokine secretion and the inflammatory response.
Authors’ contributions
Contribution to conception and design: JK, ACP, ECN, CC. Performed the experiments: AFF, MB. Analysis of the data: AFF PCT ECN CC. Contributed reagents materials analysis tools: CWP, BMI, BS, HTLW, LGN, ADMB, PB, FD, AS, ED, JAMN, MH, MS, AF, VR, ACP. Wrote the paper: ECN, CC. Review the drafts: ECN, CC, AFF, LRPF. All authors read and approved the final manuscript.