Download Association between human African trypanosomiasis and the IL6

Infection, Genetics and Evolution 7 (2007) 60–68
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Association between human African trypanosomiasis and
the IL6 gene in a Congolese population
David Courtin a,*, Jacqueline Milet a, Vincent Jamonneau b, Claude Sese Yeminanga c,
Victor Kande Betu Kumeso c, Constantin Miaka Mia Bilengue d, Christine Betard e, André Garcia f
a
Institut de Recherche pour le Développement (IRD), Unité de recherche 010: Santé de la mère et de l’enfant en milieu tropical,
Faculté de pharmacie, 4 Avenue de l’observatoire, 75270 Paris, France
b
Institut de Recherche pour le Développement (IRD), Unité de recherche 177: relations hôtes vecteurs parasites dans les trypanosomoses,
Campus International de Baillarguet, TA207/G, 34398 Montpellier Cedex 5, France
c
Programme National de Lutte contre la Trypanosomiase Humaine Africaine, s/c Ministère de la Santé, Boulevard du 30 juin no. 4310,
Commune de la Gombe, BP 3088, Kinshasa, République Démocratique du Congo
d
Ministère de la Santé, Boulevard du 30 juin no. 4310, Commune de la Gombe, BP 3088, Kinshasa, République Démocratique du Congo
e
Centre National de Génotypage, 2 rue Gaston Crémieux, BP 5721, 91057 Evry, France
f
Institut de Recherche pour le Développement (IRD), Unité de recherche 010:
Santé de la mère et de l’enfant en milieu tropical, 08 BP 841 Cotonou, Benin
Received 9 January 2006; received in revised form 31 March 2006; accepted 1 April 2006
Available online 23 May 2006
Abstract
Despite the importance of behavioural and environmental risk factors, there are arguments consistent with the existence of a genetic
susceptibility to human African trypanosomiasis (HAT). A candidate gene association study was conducted in the Democratic Republic of Congo
using a family-based sample which included a total of 353 subjects (86 trios; one case and parents (n = 258) and 23 families with more than one
case and parents (n = 95)). Polymorphisms located on the IL1a, IL4, IL6, IL8, IL10, TNFa and IFNg genes were genotyped after re-sequencing of
the genes for extensive SNP search. The T allele of the IL64339 SNP was significantly associated with a decreased risk of developing the disease
( p = 0.0006) and a suggestive association was observed for the IL1a5417 T SNP and an increased risk of developing the disease. These results
suggest that genetic variability of the IL6 and to a lesser extent the IL1a gene are involved in the development of HAT. For the TNFa and IL10 gene
polymorphisms, association results obtained here were different from those we observed in another population living under different epidemiologic
conditions. This underlines the complexity of the interactions existing between host genetic polymorphisms, parasite diversity and behavioural and
environmental risk factors in HAT.
# 2006 Elsevier B.V. All rights reserved.
Keywords: Association study; FBAT; Interleukin; Trypanosoma brucei gambiense; Human African trypanosomiasis; Human genetics; Human susceptibility
1. Introduction
Human African trypanosomiasis (HAT), or sleeping sickness, occurs in two classical forms: the chronic form caused by
Trypanosoma brucei gambiense (Tbg) in western and central
Africa and the acute form caused by Trypanosoma brucei
rhodesiense (Tbr) in eastern Africa. Chronic infection
classically lasts for years, whilst acute infection lasts only
for weeks or months. After inoculation, parasites first grow in
* Corresponding author. Tel.: +33 1 53 73 96 21; fax: +33 1 53 73 96 17.
E-mail address: [email protected] (D. Courtin).
1567-1348/$ – see front matter # 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.meegid.2006.04.001
blood and lymph (the first period or haemolymphatic stage),
and then cross the blood–brain barrier (BBB) to reach the
central nervous system (CNS) (the second period or meningoencephalitic stage). Without treatment, the disease outcome is
fatal. HAT diagnosis cannot be based only on clinical
presentation and imply the detection of the parasite by means
of biological methods. A first group of methods (serological
tests) is based on the detection of antibodies against
trypanosomes in peripheral blood and is completed by a
second group of methods (parasitological tests) relying on the
finding of trypanosomes in blood, lymphatic juice or
cerebrospinal fluid (CSF). Only subjects with positive serology
and parasitology are diagnosed as HAT cases and treated
D. Courtin et al. / Infection, Genetics and Evolution 7 (2007) 60–68
(WHO, 1998). Apart from the Tbg classical form, diversity in
clinical progression has been observed, from very chronic and
asymptomatic forms (Jamonneau et al., 2000) to acute forms
(Truc et al., 1997). The respective roles of either the virulence
of the parasite or the host susceptibility in this clinical diversity
remain unclear (Jamonneau et al., 2002).
Experimental models of infection in mice indicate that
African trypanosomes trigger potent inflammatory responses,
and it has been suggested that survival is determined by the
ability of different inbred strains to regulate inflammatory
pathology (Namangala et al., 2001). Genetic factors
implicated in the control of immunity could be involved in
both the control of infection levels and the mortality rates, as
clearly shown for Trypanosoma congolense infections in
experimental models (Kemp et al., 1997; Iraqi et al., 2000). In
these studies, the authors identified chromosomal regions
determining resistance to T. congolense infection on murine
chromosomes 1, 5 and 17. The interleukin (IL)10 and tumor
necrosis factor (TNF)a genes are located within two of these
three identified chromosomal regions, respectively, on
chromosomes 1 and 17 of the mouse. Considering the involvement of both TNFa and IL10 in the physiopathology of
HAT (cf. bellow), this result represents an additional
argument consistent with the importance of these genes
and making them possible candidate genes for the control of
trypanosome infections.
In humans, indirect arguments exist for individual susceptibility such as familial aggregation, although the role of a
shared environment has been put forward (Okia et al., 1994;
Khonde et al., 1995). More recently, epidemiological observations have allowed us to suggest the potential role of genetic
factors involved in the control of immunity, in the diversity of
clinical presentations and the progression of HAT (Jamonneau
et al., 2000; Garcia et al., 2000, 2002). Indirect arguments also
come from immunological studies conducted in human
populations. Among the effectors of the immune responses
that are likely to play an important role in the pathogenesis of
the disease, cytokines such as IL6, IL8, IL10, TNFa and
interferon (IFN)g have already been described (OkomoAssoumou et al., 1995; Rhind et al., 1997; MacLean et al.,
2001, 2004; Lejon et al., 2002; Courtin et al., 2006a).
Concerning IL6, IL8 and IL10, the effect of HAT treatment on
their levels in both CSF and plasma strengthen their
importance, although their roles remain unclear (Lejon et al.,
2002). Chronic exposure to the disease seems to disrupt the
normal physiologic equilibrium between TNFa and IL10,
consistent with the fact that TNFa and IL10 seem to be either
beneficial or detrimental (for a review, see Dumas et al., 1999).
Concerning IFNg MacLean et al. (2004) have shown that in
plasma, IFNg levels significantly decreased during the second
period of the disease. The crossing of the BBB and the invasion
of CNS by trypanosomes is a complex phenomenon involving
IL1, IL6 and TNFa (De Vries et al., 1996; Lejon et al., 2002;
Girard et al., 2005).
There is now cumulative evidence that polymorphisms of
genes involved in the control of immune response could play a
role in HAT (MacLean et al., 2004; Courtin et al., 2006b).
61
Genetic investigation of cytokine genes could then provide a
better understanding of the underlying immunological mechanisms involved in HAT pathogenesis, leading ultimately to more
effective therapeutic and prophylactic control modalities.
Recently, we conducted an association study in Côte d’Ivoire
(IC) showing that TNFa308 G/A single nucleotide polymorphism (SNP) was associated with a higher risk of developing the
disease, whereas the IL10592 C/A SNP was associated with a
lower risk (Courtin et al., 2006b).
In the investigation reported herein, our aim was to study the
role of single nucleotide polymorphisms of TNFa, IL10, IL1a,
IL4, IL6, IL8 and IFNg genes on susceptibility/resistance to
HAT by means of a family-based association study conducted
on a different population (all individuals were native from the
area and belonged to the same ethnic group in the present study)
living under different exposure conditions (prevalence of HAT
around 1% in IC (Laveissière et al., 2003)) and around 15% in
some villages of the Bandundu focus (National Control
Program, personal communication).
2. Materials and methods
2.1. Population and study design
The study took place in Bandundu province of the
Democratic Republic of Congo (DRC). All patients enrolled
in this study signed an informed consent form and the protocol
was approved by local traditional authorities (chief and village
committee) and by the local ethical committee of DRC (Public
Health Ministry).
HAT patients included in the study were diagnosed during
active and passive surveys conducted by the National Control
Programme (NCP), using their classical detection protocol. For
serology, the Card Agglutination Test for Trypanosomiasis
(CATT) (Magnus et al., 1978) was performed on whole blood,
and trypanosomes were detected in positive CATT individuals,
using direct microscopic examination of blood or lymphatic
juice when swollen lymph nodes were present. Cases are
defined as subjects presenting both serological and parasitological positive tests.
Basically, an association study compares the frequencies of
genetic polymorphisms between cases and controls. To avoid
population admixture that can lead to false associations, familybased association methods have been developed such as the
transmission disequilibrium test (TDT) (Spielman et al., 1993).
The sampling units in these methods consist of an affected child
and his parents or unaffected siblings. Extensions are now
available which able to include a larger type of sample units
including nuclear family with several affected children. Our
population study consisted in 353 individuals, 135 HAT cases
and 218 related controls. This group of 353 individuals was
constituted by 86 trios composed of one case and parents
(n = 258), 20 nuclear families with two cases and parents
(n = 80) and 3 nuclear families with three cases and parents
(n = 15). All individuals, from the Yansi ethnic group, were
born in the area and had been exposed to the risk of infection
since their birth.
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D. Courtin et al. / Infection, Genetics and Evolution 7 (2007) 60–68
Table 1
Primers and probes used in the TaqMan PCR assay
SNP
A1/A2 Primer name
Primer
RD
IL1a 889
C/T
IL1a 2097
Probe
RQ
IL1a 889F
IL1a 889R
GGCCACAGGAATTATAAAAGCTGAGA
GGGAGAAAGGAAGGCATGGATTTT
VIC CCTTCAATGGTGTTGCC
FAM CCTTCAATGATGTTGCC
NFQ
NFQ
C/T
IL1a 2097F
IL1a 2097R
CGCTTCACCAGGTGTCTGT
TCTAACCTCTGATGCTGGTGTCA
VIC AACACATCACGTTAGGAG
FAM AACACATCACATTAGGAG
NFQ
NFQ
IL1a 4716
A/C
IL1a 4716F
IL1a 4716R
TGGGTGATTTCACTTCTCTTTGCTT
CACAGTCTAGTACAAACAGGGAAAATAGT
VIC TCTGGATTGGAATATT
FAM ATCTGGATTGGCATATT
NFQ
NFQ
IL1a 5417
C/T
IL1a 5417F
IL1a 5417R
AGTCTGTTGATCAAACTCACAAGTAACA
GGAGGTTTTGCCTCACAAATATGTT
VIC ATAAAGATCTTCCTGGTTTGG
FAM ATAAAGATCTTCCTAGTTTGG
NFQ
NFQ
IL4 33
T/C
IL4 33F
IL4 33R
ATTGCATCGTTAGCTTCTCCTGAT
ACCCATTAATAGGTGTCGATTTGCA
VIC ACAATGTGAGACAATTA
FAM ACAATGTGAGGCAATTA
NFQ
NFQ
IL4 110
A/G
IL4 110F
IL4 110R
GTCTCACCTCCCAACTGCTT
CTGTAAGGTGATATCGCACTTGTGT
VIC TTCCTGCTAGCATGTG
FAM CCTGCTGGCATGTG
NFQ
NFQ
IL4 8492
C/A
IL4 8492F
IL4 8492R
AGTGCCACAGTAGGCTTGATC
CTCTGGTTGGCTTCCTTCACA
VIC TCTGCAAAAGAAACATT
FAM TTCTGCAAAATAAACATT
NFQ
NFQ
IL6 320
C/T
IL6 320F
IL6 320R
GGGCTGCTCCTGGTGTT
GGCGGCTACATCTTTGGAATCTT
VIC CCCAGTACCCCCAGGAG
FAM CCCAGTACCCTCAGGAG
NFQ
NFQ
IL6 1890
G/T
IL6 1890F
IL6 1890R
GTCAAATGTTTAAAACTCCCACAGGTT
GCAGCCAGAGAGGGAAAAGG
VIC CCCTGCGAGTACCTT
FAM CCCTGAGAGTACCTT
NFQ
NFQ
IL6 4339
C/T
IL6 4339F
IL6 4339R
TCATCTCATTCTGCGCAGCTTTA
CCATGCTACATTTGCCGAAGAG
VIC CTGCAGGAACTCCT
FAM CTGCAGAAACTCCT
NFQ
NFQ
IL8 251
A/T
IL8 251F
IL8 251R
TCTGTCACATGGTACTATGATAAAGTTATCTAGAAAT
CGGAGTATGACGAAAGTTTTCTTTGATC
VIC AAGCATACAATTGATAATT
FAM AGCATACATTTGATAATT
NFQ
NFQ
IL8 102
T/C
IL8 102F
IL8 102R
GACAAGAGCCAGGAAGAAACCA
CAGGAAGGCTGCCAAGAGA
VIC CTTGGAAGTCATATTTACA
FAM TTGGAAGTCATGTTTACA
NFQ
NFQ
IL8 396
G/T
IL8 396F
IL8 396R
CACTTAGGAAAGTATAAAGGTTTGATCAATATAGATATTCTG VIC AAATATATGCATGCTACCTGGTAT NFQ
ACCGTGGTTCTCAATAGGACATACTA
FAM ATGCATGCTACATGGTAT
NFQ
IL10 592
C/A
IL10 592F
IL10 592R
GGTAAAGGAGCCTGGAACACATC
GCCCTTCCATTTTACTTTCCAGAGA
VIC CCCGCCTGTCCTGTAG
FAM CCGCCTGTACTGTAG
NFQ
NFQ
IL10 435
C/T
IL10 435F
IL10 435R
ATGATACAGTAAATGTGCAGGAAACCT
CGCCAGCAGGATCTTATAAGTTTCT
VIC CACGAGAGAGAACG
FAM CACGAGAAAGAACG
NFQ
NFQ
TNFa 863 C/A
TNFa 863F GTAGGAGAATGTCCAGGGCTATG
TNFa 863R CCCTCTACATGGCCCTGTCT
VIC ACCCCCCCTTAACG
FAM CCCCCACTTAACG
NFQ
NFQ
TNFa 376 G/A
TNFa 376F CCCCTCCCAGTTCTAGTTCTATCTT
TNFa 376R CCTCAAAACCTATTGCCTCCATTTC
VIC CTGTCTGGAAGTTAGAAG
FAM CTGTCTGGAAATTAGAAG
NFQ
NFQ
TNFa 308 G/A
TNFa 308F CCAAAAGAAATGGAGGCAATAGGTT
TNFa 308R GGACCCTGGAGGCTGAAC
VIC CCCGTCCCCATGCC
FAM CCCGTCCTCATGCC
NFQ
NFQ
IFNg 183
G/T
IFNg 183F
IFNg 183R
GGGCATAATGGGTCTGTCTCAT
GGCATTTGGGTGTTGTAGTTAGAGT
VIC CTTGGGTCCTTTGACG
FAM TTGGGTCATTTGACG
NFQ
NFQ
IFNg 4565
C/T
IFNg 4565F
IFNg 4565R
GACTCATCAATCAAATAAGTATTTATAATAGCAACTTTTGT
CACATAGCCTTGCCTAATTAGTCAGA
VIC ACAGTCACAGGATATAG
FAM CAGTCACAAGATATAG
NFQ
NFQ
IFNg 5003
C/T
IFNg 5003F
IFNg 5003R
ACAACTACTTATGCTGTGTTGGACTT
GAAGACTCCCCTCCCTACTAATTCA
VIC TCACTCCAGGTCTCAC
FAM CACTCCAGATCTCAC
NFQ
NFQ
IFNg 5295
A/G
IFNg 5295F
IFNg 5295R
TTCTAGCCCCTTCTCCACCTT
AGCATTGGATGAGGGAGAGGAA
VIC CTCCTTCATTTCAGAATC
FAM CCTTCGTTTCAGAATC
NFQ
NFQ
A1/A2, allele 1/allele2. RD, reporter dye at the 50 end of each probe (VIC dye is linked to the allele 1 probe and FAM dye linked to the allele 2 probe). RQ, reporter
quencher at the 30 end of the probe; NFQ, non-fluorescent quencher.
2.2. SNP discovery
SNP discovery using re-sequencing (Vasilescu et al., 2003)
was performed to detect IL1a, IL4, IL6, IL8, IL10, TNFa and
IFNg gene polymorphisms in 16 healthy individuals and 16
affected individuals, randomly selected, among those included
in our study. DNAs from the coding sequence, exon flanking
regions and promoter regions of these genes were amplified by
D. Courtin et al. / Infection, Genetics and Evolution 7 (2007) 60–68
PCR. The PCRs were carried out in a 15 ml reaction mixture
containing 25 ng of DNA. The list of each gene’s primer is
available on the web site of the Centre National de Génotypage
(CNG), France (www.cng.fr). DNA amplified fragments were
purified with Bio-Gel P-100 (Bio-Rad1) and sequencing
reactions were achieved using BigDye Terminator Mix
(Applied Biosystems1). The reaction solutions were purified
using G50 Sephadex resin (Amersham Pharmacia Biotech AB)
and subsequently analysed using an ABI PRISM 3700 DNA
Analyser (Applied Biosystems1). We compared sequences
using Genalys software developed by CNG (Takahashi et al.,
2002) in order to identify SNPs and estimate their frequency in
our study population.
2.3. SNP genotyping
SNPs were genotyped using TaqMan primers and probes
(Applied Biosystems1). The allelic discrimination was based
on the design of two TaqMan probes, specific for the wildtype
allele and the mutant allele. One allelic probe was labelled with
the FAM dye and the other with the fluorescent VIC dye. The
sequences of the primers and allele-specific probes used in the
TaqMan assays are shown in Table 1. The TaqMan PCR
reactions were done according to the instructions provided with
the kit using 3 ng of genomic DNA and were analysed in a
7900HT Sequence Detection System (Applied Biosystems1)
with Applied Biosystems1 Genotyper software (SDS system,
Version 2.0).
63
3. Results
The mean age (range) of HAT cases in the population study
was 14.69 (2–39) years. The sex ratio (male:female) was 0.65
(53/82).
Ninety-nine SNPs were identified after re-sequencing the
candidate genes in individuals from the Congolese population:
32 in IL1a, 5 in IL4, 17 in IL6, 10 in IL8, 9 in IL10, 16 in TNFa
and 10 in IFNg (Fig. 1). Among these 99 SNPs, 22 were
selected for larger-scale genotyping based on location (SNPs in
promotor and exons were preferred), linkage disequilibrium
(SNPs that were not in complete LD with each other were
selected), and allele frequencies greater than 5% except for
TNFa308 G/A (Fig. 1). For this last SNP, the allele frequency
was 3%, but its potential involvement in susceptibility/
resistance to the disease argued for keeping it in the analysis.
Information on selected SNPs is shown in Table 2.
Statistical analysis revealed that all studied polymorphisms
were in Hardy–Weinberg equilibrium in our population sample.
The family-based association study results are shown in
Table 3. Nominal p-values (not adjusted for multiple testing)
<0.05 were found for three SNPs: IL64339 C/T, IL1a5417 C/T and
IL10592 C/A. Under a dominant model, the IL64339 T allele was
less transmitted from heterozygote parents to affected offspring
( p = 0.0006), consistent with a protective effect. The IL1a5417
C/T polymorphism showed an excess transmission ( p = 0.009)
of the rare allele from parents to affected offspring, suggesting
an increased risk of developing the disease. A weak trend
( p = 0.043) was observed for an association between the
2.4. Statistical analysis
The Spearman Chi-square test was used to compare the
frequencies of observed and expected genotypes under Hardy–
Weinberg equilibrium, using STATA software (StataCorp 1999,
Release 6.0). Haplotypes and their frequencies were estimated
with the EM algorithm (Laird, 1993).
We tested for an association of individual SNPs with HAT
using the family-based association test (FBAT) programme
(Horvath et al., 2001), and for haplotypes, using the haplotype
extension (HBAT) of the FBAT programme (Horvath et al.,
2004). The bi-allelic mode was used to test each haplotype
(specific haplotype) and the multiallelic mode was used to test
the global haplotype. The global haplotype tests all haplotypes
simultaneously and computes a large sample Chi-square
statistic. To ensure its validity as a test of association in the
pedigree, the FBAT statistic was calculated using empirical
variance (the – e option) in single-locus and haplotype analyses.
As we have no information about the relevant genetic model,
the three possible genetic models were used for analysis:
additive, dominant and recessive (only results for the model
with the lowest p-value was presented here). Then, we took into
account for the adjustment of the significance level the fact that
we tested three models for each polymorphism. As 22
polymorphisms were tested, adjusting for multiple testing by
Bonferroni correction means that the association for a single
marker has to be significant at a = 0.05/(22 3) = 0.0008 to
achieve an overall significance of 0.05.
Table 2
Results of SNP discovery and information on the SNPs selected for testing
association
Genes
Chromosome
location
Detected
SNPs
Selected
SNPs
SNPs
location
Amino acid
change
IL1a
2q14
32
889
2097
4716
5417
Promotor
Intron 3
Intron 4
Intron 5
–
–
–
–
IL4
5q31.1
5
33
110
8492
Exon 1
Exon 1
Intron 3
50 UTR
Leu15Leu
–
IL6
7p21
17
320
1890
4339
Exon 2
Intron 3
Exon 5
Ser32Pro
–
Phe201Phe
IL8
4q12-q13
10
251
102
396
Promotor
Exon 1
Intron 1
–
50 UTR
–
IL10
1q31-q32
9
592
435
Promotor
Intron 1
–
–
TNFa
6p21.3
16
863
376
308
Promotor
Promotor
Promotor
–
–
–
IFNg
12q14
10
183
4565
5003
5295
Promotor
Exon 4
Intron 4
Intron 4
–
30 UTR
–
–
64
D. Courtin et al. / Infection, Genetics and Evolution 7 (2007) 60–68
Fig. 1. Gene maps and haplotypes of the seven cytokine genes investigated in the study. Coding exons are marked by black blocks, and 50 and 30 UTRs by white
blocks. Asterisks indicate polymorphisms genotyped in a larger population. The regions that have been sequenced are shown by a horizontal line below each gene.
Haplotypes with frequencies >0.05 (excepted for TNFa) are shown for all SNPs. A1 and A2 corresponded to the frequent and rare allele, respectively.
D. Courtin et al. / Infection, Genetics and Evolution 7 (2007) 60–68
65
Table 3
Family-based association test results
Gene
SNP
Alleles
Fq
No. of familiesa
Modelb
Observed valuec
Expected valued
IL1a
889
T
C
0.413
0.587
37
d
30.000
28.000
0.603
0.546
2097
T
C
0.373
0.627
50
d
31.000
33.056
0.543
0.587
4716
C
A
0.295
0.705
42
d
24.000
25.917
0.529
0.596
5417
T
C
0.194
0.806
37
d
35.000
25.333
2.612
0.009
33
C
T
0.515
0.485
44
a
54.000
50.000
0.929
0.353
110
G
A
0.169
0.831
35
a
29.000
22.500
1.786
0.074
8492
A
C
0.439
0.561
47
a
45.000
50.000
1.211
0.226
320
T
C
0.066
0.934
20
d
16.000
12.500
1.460
0.144
1890
T
G
0.318
0.682
55
d
34.000
38.658
1.160
0.245
4339
T
C
0.143
0.857
34
d
10.000
20.750
3.410
0.0006
251
T
A
0.159
0.841
4
r
2.000
1.778
0.183
0.855
102
T
C
0.242
0.758
19
r
16.000
18.167
0.686
0.492
396
T
G
0.508
0.492
34
r
28.000
23.000
1.873
0.061
592
A
C
0.451
0.549
45
d
41.000
33.444
2.021
0.043
435
T
C
0.293
0.707
41
d
29.000
28.778
0.065
0.948
863
A
C
0.098
0.902
24
a
14.000
18.333
1.363
0.173
376
A
G
0.047
0.953
9
a
5.000
6.500
0.655
0.512
308
A
G
0.154
0.846
25
a
19.000
18.167
0.288
0.773
183
T
G
0.040
0.960
7
a
3.000
4.000
0.632
0.527
4565
T
C
0.085
0.915
20
a
9.000
11.500
0.928
0.353
5003
T
C
0.051
0.949
11
a
7.000
6.500
0.243
0.808
5295
G
A
0.140
0.860
32
a
22.000
24.667
0.783
0.433
IL4
IL6
IL8
IL10
TNFa
IFNg
a
pe
Z
Number of informative families (i.e. families with at least one heterozygous parent).
Genetic model (a = additive, d = dominant and r = recessive). The three genetic models were tested for each SNPs, only the more relevant one was presented.
c
Number of transmitted alleles observed.
d
Expected value of transmitted alleles under the null hypothesis.
e
Nominal p-value; in bold = p-value remaining significant after Bonferroni correction; significance level retained after correction (66 tests (22 SNPs 3 models)):
0.0008.
b
66
D. Courtin et al. / Infection, Genetics and Evolution 7 (2007) 60–68
Table 4
Genetic association between IL6 haplotype and HAT
Gene
IL6
Haplotype a
H1:
H2:
H3:
H4:
CGC
CTC
CTT
TGC
Frequencyb
0.636
0.175
0.125
0.064
Global haplotypef FBAT
Specific haplotype FBAT
No. of familiesc
Observed valued
Expected valuee
Z
p
x2(d.f.)
p
24
34
31
19
78.000
37.000
15.000
19.000
75.250
32.667
23.167
16.000
1.026
1.371
2.726
1.279
0.305
0.170
0.006
0.201
10.243 (4)
0.036
a
These haplotypes correspond to the combination of the SNPs listed in Table 3: 320C/T, 1890G/T, 4339C/T for the IL6 gene. The polymorphism 4339C/T (in bold in
haplotype column) is the one previously found significantly associated.
b
EM estimates haplotype frequency.
c
Number of informative families (i.e. families with at least one heterozygous parent).
d
Number of transmitted haplotypes observed.
e
Expected value of transmitted haplotypes under the null hypothesis.
f
Global haplotype tests all haplotypes simultaneously and computes a large sample Chi-square statistic.
IL10592 C/A polymorphism and an increased risk of HAT.
However, the only association that remained significant after
Bonferroni correction concerned the IL64339 C/T SNP. No
association between all other polymorphisms and the disease
was found, including TNFa308 G/A, which we found associated
with a higher risk of developing the disease in IC.
Haplotypes were analysed and a statistically significant
association was found between IL6 H3 and the disease
( p = 0.034 using a global haplotypic test). This haplotype
corresponds to the combination of 320C/T, 1890G/T and 4339C/T
for the IL6 gene. Results are summarized in Table 4. The IL6 H3
haplotype presented a frequency of 0.125 and was less
transmitted from parents to affected offspring than expected
under the null hypothesis of absence of association ( p = 0.006).
This haplotype is the only one harbouring the T allele at the
IL64339 locus, consistent with the results obtained with the
IL64339 T allele alone. For the IL1a, IL4, IL8, IL10, TNFa and
IFNg genes, no haplotype was significantly associated with the
disease.
4. Discussion
In this study, 22 SNPs in seven cytokine genes were
investigated to test genetic association with HAT in a
Congolese population. Our main finding is that the IL64339 T
allele is significantly associated with a lower risk of developing
the disease. The trend obtained with the IL1a5417 C/T
polymorphism, and to a lesser extent IL10592 C/A, must be
considered with interest but needs further confirmation in other
populations.
The IL64339 C/T polymorphism, located in exon 5 of the IL6
gene is synonymous. However, this polymorphism may be in
linkage disequilibrium with another polymorphism located in
the part of IL6 gene which was not sequenced or in a nearby
gene. This polymorphism can also affect gene function by
altering the stability, splicing or location of mRNA (Cartegni
et al., 2002; Tabor et al., 2002) and can therefore be considered
as an interesting candidate for association (Risch, 2000).
The role that IL6 could play in HAT strengthens the
pertinence of our finding. A crucial step in HAT is the invasion
of CNS by the parasites when they cross the BBB. Although the
mechanisms remain unclear, a hypothesis could be that the
modification of the BBB permeability stems from the presence
of inflammatory mediators, such as IL1, IL6 and TNFa, in
blood and/or in CNS (De Vries et al., 1996). Girard et al. (2005)
demonstrated in vitro that Tbg induced the synthesis of IL6 by
human bone marrow endothelial cells, and Lejon et al. (2002)
confirmed the involvement of this cytokine whose level
increases in CSF during Tbg infection and decreases after
treatment. These findings corroborate with data from Trypanosoma brucei brucei-infected mice, where the detection of IL6
in the brain correlates with astrocyte activation (Hunter et al.,
1992) in the choroid plexus (Quan et al., 1999). However, the
role of this cytokine could be much more complex and IL6 is
certainly involved in other steps than neuropathogenesis during
disease progression. In a sub-sample of the same population, we
showed that IL6 serum concentrations in HAT cases were
higher than in both controls and seropositive individuals
without parasitological confirmation (Courtin et al., 2006a).
The same differences between cases and controls were shown
in serum concentration by Lejon et al. (2002), but in this case,
the serum level did not decrease after treatment. All these
results emphasize the potential role of this cytokine in the
pathogenesis of HAT and the necessity to confirm our results in
another population. Unfortunately, the recent events in Côte
d’Ivoire did not allow us to proceed with this analysis in our
previous population for which DNA is no longer available.
To our knowledge, the IL1a5417 C/T polymorphism, located
in intron 5 of the IL1a gene has not been associated with a
functional effect. However, in this case also, this polymorphism
may be in linkage disequilibrium with another unidentified
coding polymorphism in the gene or in a nearby gene. IL1
participates in macrophage activation during early infection in
mice (Sternberg, 2004) and plays a key role in the recruitment
of leukocytes into the CNS through the BBB during CNS
infection (Borges, 1992; Ching et al., 2005). Its possible
association with the HAT variable risk must be confirmed.
We showed previously in IC that individuals who are
homozygous for the TNFa308 A allele have a higher risk of
developing the disease (Courtin et al., 2006b). This effect was
D. Courtin et al. / Infection, Genetics and Evolution 7 (2007) 60–68
not found in the present study. The same pattern of discrepant
results has already been shown for the same polymorphism and
cerebral malaria in different populations (McGuire et al., 1994).
The conclusions concerning the role of TNFa308 G/A seemed
complex because of the low allele frequencies that make it
difficult to assess the effect of homozygosity (Knight et al.,
1999). The same pattern of differences in allele frequencies
exists between our two populations (i.e. DRC and IC) and could
be responsible for the discrepancy observed between our
studies. In DRC, only seven subjects (representing 2% of the
population) were homozygous for TNFa308 AA genotype,
whereas they represented 6% of the population in IC. Although
a lack of power could explain the heterogeneity of the results,
this difference may result from the complexity of the
interactions between environmental and genetic risk factors,
as well as of the heterogeneity of genetic control in infections.
The result obtained for the IL10592 C/A polymorphism was
not significant. However, it is interesting to note that this trend
could be consistent with a higher risk of developing the disease,
whereas in IC, the same promoter polymorphism was
significantly associated with a lower risk of HAT (Courtin
et al., 2006b). Although experimental evidence points to a
potentially protective role of IL10 in the late stage of the disease
by countering the important production of TNFa, excessive
down-regulation of TNFa might be deleterious (Blackwell and
Christman, 1996). Another explanation could be that the
polymorphism studied (i.e. IL10592 C/A) has no effect per se
but is in linkage disequilibrium with another unidentified
coding polymorphism in the gene in IC. Variation of this
linkage disequilibrium across our populations might lead to
different findings (Tu and Whittemore, 1999).
Heterogeneity of the populations and of the definition of
phenotypes must also be taken into account (Palmer and
Cookson, 2000). In IC, the prevalence of the disease was
lower than 1% and our population lived in a sub-urban area
and was composed of 56% migrants from Sudanese areas
where HAT was absent. In some villages of the Bandundu
area or DRC, the prevalence was greater than 15% (National
Control Programme, personal communication) and the entire
population studied belonged to the same ethnic group and
lived in rural conditions, exposed to the risk of HAT since
their birth. Such differences can influence the development of
the immune system and can lead to dissimilar interactions
between human host and parasites, as already described for
HAT (Jamonneau et al., 2002; Garcia et al., 2002) as well as
for malaria (Baird, 1995).
Phenotypic heterogeneity can be directly related to the
complexity of HAT (Jamonneau et al., 2002) and of its
progression (Garcia et al., 2000). However, as all individuals
harbouring parasites receive an immediate treatment whatever
their clinical presentation, the variability in severity of infection
cannot be taken into account. The main consequence could be a
phenotypic heterogeneity between, but also within, our two
populations (i.e. IC and DRC).
In conclusion, this study confirms that differing host
responses to Trypanosoma infections may result from immune
response polymorphisms in host populations (MacLean et al.,
67
2004; Sternberg, 2004). Further studies must be conducted with
larger populations to confirm our results and to determine a
potential immunogenetic component for the disease progression and outcome in HAT.
Acknowledgments
We are very grateful to the population included in the study
and to the HAT National Control Programme of DRC. We
acknowledge Philippe Büscher and Alexandre Vasilescu for
technical assistance and helpful comments on the manuscript.
This study received financial support from: Institut de
Médecine et Epidémiologie Africaine (IMEA, France),
Direction Générale à l’Armement (DGA, France), Fondation
des Treilles (France), Centre National du Génotypage (CNG,
France) and Institut de Recherche pour le Développement
(IRD, France).
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