a stat6 gene polymorphism is associated with high infection levels in urinary schistosomiasis

ORIGINAL ARTICLE

A STAT6 gene polymorphism is associated with highinfection levels in urinary schistosomiasis

H He1,2, A Isnard1,2, B Kouriba3, S Cabantous1,2, A Dessein1,2, O Doumbo3 and C Chevillard1,2

1INSERM, U399, Marseille, France; 2Laboratory of Parasitology Mycology, Faculty of Medicine la Timone, University of Aix-Marseille,Marseille, France and 3Department of Epidemiology and Parasitic Diseases, Faculty of Medicine, Pharmacy and Odonto-Stomatology,Bamako, Mali

Th2-mediated immunity is critical for human defence against schistosome, and susceptibility to infection is controlled by amajor genetic locus, mapped on the 5q31–q33 region comprising the genes IL4, IL5 and IL13. We have reported an associationbetween the rs1800925 polymorphism in the IL13 promoter and infection levels in a Dogon population (693 subjects in Segueand 148 in Boul), where Schistosoma haematobium is endemic. In the same population, we investigated whether otherpolymorphisms in genes involved in type 2 cytokine immune response could affect susceptibility to schistosome infection. Bylogistic regression analysis, we found an association between a single-nucleotide polymorphism (SNP) in the STAT6 gene(rs324013) and infection levels (P¼ 0.04). We confirmed this association in analyses restricted to subjects under 20 years ageand living in Boul, the village with the highest levels of infection (P¼ 0.005). We detected an additive effect of the rs324013 andrs1800925 polymorphisms (P¼ 0.011). These SNPs were not strongly correlated with any other tested markers surroundingthe two genes. Furthermore, electrophoretic mobility shift assay has shown that both polymorphisms affect transcription factorbinding. These results are consistent with the Th2 cytokine pathway enhancing resistance to schistosome infection in humans.Genes and Immunity advance online publication, 14 February 2008; doi:10.1038/gene.2008.2

Keywords: Th1/Th2 cells; parasitic-helminth; human; molecular biology

Introduction

Schistosomiasis is the fourth most prevalent disease inthe world and the second most prevalent parasiticdisease after malaria.1 It comprises a group of chronicdiseases caused by helminth digenetic trematode of theSchistosoma genus. Schistosoma mansoni and Schistosomahaematobium inhabit mesenteric and vesical venousplexus, respectively, where female worms lay anaverage of 300–3000 eggs per day. The eggs cause majorpathological disorders; they may pass into faeces(S. mansoni) or urine (S. haematobium) or remain trappedwithin the tissues (mainly the liver or bladder and ureterwalls). S. mansoni causes hepatic fibrosis associatedwith portal blood hypertension, which is often lethal.S. haematobium causes obstructive nephropathy thatcan be aggravated by urinary bacterial infections.2–4 Inendemic regions, 5–20% of the population is affected bysevere schistosomiasis; the annual global mortality rate is250 000–300 000.5

Studies of the prevalence and intensity of infection byS. mansoni in a Brazilian population living in an endemicarea, have shown a heterogeneous distribution of

infection levels.6 Exposure, age and gender explain onlyone-third of the variance of infection levels withinthis population, suggesting that other factors wereinvolved.6–8 Individuals with the highest infection levelswere grouped within families, rather than being ran-domly distributed. Further analysis showed that infec-tion levels depended on a major codominant locus (SM1)that accounted for half the variance of infection levels.8

This major locus was mapped on 5q31–q33,9,10 a regioncontaining the Th2 gene cluster (including the IL4, IL5and IL13 genes).11–13 This result was further confirmed byan independent study in Senegal.14 Furthermore, we andothers have shown that sterile immunity in humanschistosomiasis is dependent on IgE levels and eosino-phils,15–17 and on the Th1/Th2 balance.18,19

We have obtained similar results from studies basedon a S. haematobium-infected population. Indeed, highinfection levels were also clustered in families.20

Moreover, immune responses involving IgE are stronglyassociated with protection against S. haematobium.21

Thus, we tested whether allelic variants in the5q31–q33 region would predispose to high levels of S.haematobium infection. Polymorphisms in the IL13 gene(rs1800925 and rs2069743) were associated with suscept-ibility to S. haematobium infection.20 In particular, subjectscarrying the rs1800925T/T genotype had a lower level ofinfection than individuals with rs1800925 C/C or C/Tgenotypes.

Similarly, single-nucleotide polymorphisms (SNPs)have been associated with the control of infectionlevels, such as in Ascaris lumbricoides (another helminth

Received 5 September 2007; revised 3 December 2007; accepted8 January 2008

Correspondence: Dr C Chevillard, Laboratory of ParasitologyMycology, Faculty of Medicine la Timone, University of Aix-Marseille, INSERM, U399, Marseilles, 27, Boulevard Jean Moulin13385 Marseilles Cedex 5, France.E-mail: [email protected]

Genes and Immunity (2008), 1–12& 2008 Nature Publishing Group All rights reserved 1466-4879/08 $30.00

www.nature.com/gene

infection). Indeed, we recently showed that twopolymorphisms in the IL13 gene (rs1800925 andIL13þ 111C/A) are associated with Ascaris infectionlevels in African children living in an endemic area inCameroon (Eboumbou et al., personal communication).The rs1800925C allele was associated with suscepti-bility to infection, whereas the IL13þ 111C allele wasassociated with protection against Ascaris infection.Similarly, a STAT6 variant (rs324015) was associatedwith resistance to ascariasis in children from an endemicarea in China.22

These associations suggest that polymorphisms ingenes encoding major components of the Th2 pathwaycould have significant effects on S. haematobium infectionin populations from endemic areas. In this study, weinvestigated whether genetic polymorphisms in thegenes encoding major components of the Th2 immuneresponse are associated with the control of S. haematobiuminfection levels in a Malian population; it includedIL4 (main Th2 cytokine), IL4RA, IL13RA1, IL13RA2 (IL4and IL13 receptor chains), STAT6 (signal transducer) andGATA3 (main switch in determining Th1–Th2 responses).Here, we show that a STAT6 polymorphism in thepromoter (rs324013) is associated with the control ofinfection levels and has an additive effect with rs1800925,

a polymorphism previously found to have an association inthis same population. These two SNPs, which are notknown to be strongly correlated with another polymorph-ism, modify the binding of nuclear factors to the promoterregions of their respective genes.

Results

A linear regression analysis of the Segue/Boul populationsuggests that the rs324013 polymorphism in STAT6 isassociated with infection levelsWe tested putative associations between 18 SNPs ingenes encoding major components of the Th2 immuneresponse (IL4, IL4RA, IL13RA1, IL13RA2, STAT6 andGATA3) and infection levels in the Segue/Boul popula-tion. Information on the markers is given in Table 1.Subjects over 4 years age (693 from Segue and 148 fromBoul) were included in the study. A total of 480 subjectsaccepted to give blood sample for DNA extraction. SNPfrequencies are shown in Table 2. All the markers were inHardy–Weinberg equilibrium.

We tested for potential associations between indivi-dual polymorphisms and infection levels by multivariateanalysis. We performed logistic regression to analyse the

Table 1 SNPs used to characterize the Th2 pathway

Gene SNPs Position accordingto coordinate system

Sequences Frequency (HapMap) Frequency (Applied Biosystems)

CEUa CHBb JPTc YRId AfAme Caucf Ching Japnh

IL13 rs1800925 132020708 TAGGAAAA[C/T]GAGGGAAG NA NA NA NA 0.33 0.19 0.21 0.18rs2069743 132021174 ACCTGGAA[A/G]TCTGAACT NA NA NA NA NA NA NA NArs34255686 132021930 GTGATGTT[G/T]ACCAGCTCC NA NA NA NA NA NA NA NArs2066960 132022334 AGCACCAT[C/A]ATAGGCCC NA NA NA NA 0.19 0.17 0.33 0.30rs1295686 132023742 TCTACTCA[C/T]GTGCTGAC 0.24 0.31 0.28 0.28 NA NA 0.36 0.32

IL4 rs2070874 132037609 ACTAATTG[C/T]CTCACATT 0.16 0.22 0.27 0.47 0.47 0.18 0.21 0.35rs2243266 132041688 GAAAAAAC[A/G]TTAAAATA 0.16 0.23 0.27 0.37 NA 0.16 0.23 0.27rs2243291 132046882 AACACTGT[C/G]CACAGTGG 0.17 0.22 0.28 0.26 0.46 0.17 NA NA

IL4RA rs6498012 27239475 CATTTCTT[C/G]GTTGGAAA 0.30 0.46 0.22 0.24 0.30 0.43 0.48 0.26rs3024548 27262032 GCATGCAG[C/G]GTTCACCA 0.44 0.37 0.28 0.28 0.36 0.46 NA NArs8674 27283288 CTCAGCCA[C/T]CCTGTGGG NA NA NA NA NA 0.39 0.46 0.33

IL13RA1 rs5957048 117631348 GTCTTCCA[C/T]AATTTTTG 0.15 0.41 0.44 0.46 NA 0.15 0.41 0.44rs1316954 117672338 AAGCCATT[A/G]TGTTTTAG 0.17 0.41 0.45 0.47 0.47 0.11 0.47 0.45rs759147 117728470 TTCAGATA[A/C]CTGGGGCA 0.13 0.40 0.43 0.35 NA 0.13 0.40 0.43

IL13RA2 rs5946040 114070746 CGTCATTT[G/T]GGAAGCAA 0.16 0.00 0.02 0.24 NA 0.16 0.00 0.02rs10482478 114090848 AAGCCATT[A/G]TGTTTTAG 0.24 0.02 0.14 0.29 NA 0.24 0.02 0.14

STAT6 rs324010 55768531 ACGCACAG[A/G]AGAAGCCG 0.47 0.33 0.27 0.26 NA NA NA NArs324015 55776367 TGTTGGGG[C/T]GTGTCTCA 0.27 0.49 0.41 0.19 NA 0.27 0.49 0.41rs324011 55788449 CTAGGGAC[C/T]GTCCCCAC 0.40 0.36 0.16 0.07 0.21 0.41 0.19 0.12rs324013 55796928 GGAGACGA[C/T]ACAGTTGA 0.48 0.42 0.25 0.45 0.43 0.45 0.43 0.27

GATA3 rs444762 8143266 TTAGCTCC[A/C]CTGCCACC NA NA NA NA 0.41 0.27 0.28 0.41rs406103 8151627 GCAAACAG[C/T]GTCACCAC NA NA NA NA 0.30 0.33 0.24 0.37rs477461 8160149 GCTTTGCA[A/G]AGAGCCCA 0.15 0.23 0.33 0.32 0.22 0.23 NA NA

Abbreviations: IL, interleukin; SNP, single-nucleotide polymorphism.HapMap reference population: aCEPH (Utah residents with ancestry from northern and western Europe), bHan Chinese from Beijing, China,cJapanese from Tokyo, Japan and dYoruba from Ibadan, Nigeria.Applied Biosystems reference population: eAfro-American population, fCaucasian population, gChinese population, hJapanese population.All the polymorphisms were genotyped by TaqMan assay except rs1800925 and rs2069743 which were genotyped by primer extension.

STAT6 and high infection levels in schistosomiasisH He et al

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Table 2 Frequencies of the polymorphisms in the Segue/Boul population

Gene SNPs Location inthe gene

Genotype Frequency in thestudy population

Hardy–Weinbergequilibrium

test w2 (P-value)a

IL13 rs1800925 Promoter C/C 117 (25.32%) w2¼ 1.77; P¼ 0.41C/T 254 (54.98%)T/T 91 (19.70%)

rs2069743 Promoter A/A 102 (55.43%) w2¼ 0.13; P¼ 0.94A/G 69 (37.50%)G/G 13 (7.07%)

rs34255686 Exon 1 G/G 387 (81.99%) w2¼ 4.56; P¼ 0.10G/T 80 (16.95%)T/T 5 (1.06%)

rs2066960 Intron 1 C/C 313 (67.31%) w2¼ 3.69; P¼ 0.16C/A 135 (29.03%)A/A 17 (3.66%)

rs1295686 Intron 3 T/T 229 (50.44%) w2¼ 2.65; P¼ 0.27T/C 183 (40.31%)C/C 42 (9.25%)

IL4 rs2070874 Exon 1 T/T 158 (33.83%) C2 ¼ 0.58; P¼ 0.75T/C 205 (43.90%)C/C 104 (22.27%)

rs2243266 Intron 2 G/G 146 (31.33%) w2¼ 0.44; P¼ 0.80G/A 212 (45.49%)A/A 108 (23.18%)

rs2243291 30 UTR C/C 34 (50.11%) w2¼ 0.77; P¼ 0.68C/G 193 (41.33%)G/G 40 (8.56%)

IL4RA rs6498012 Promoter G/G 245 (52.24%) w2¼ 0.38; P¼ 0.83G/C 202 (43.07%)C/C 22 (4.69%)

rs3024548 Intron 3 G/G 201 (43.41%) w2¼ 1.90; P¼ 0.39G/C 223 (48.17%)C/C 39 (8.42%)

rs8674 Exon 10 C/C 153 (32.97%) w2¼ 0.45; P¼ 0.80C/T 185 (39.87%)T/T 126 (27.16%)

IL13RA1 rs5957048 Promoter T/T 210 (45.36%)T/C 117 (25.27%)C/C 136 (29.37%)

rs1316954 Intron 4 A/A 225 (48.60%)A/G 114 (24.62%)G/G 124 (26.78%)

rs759147 30 UTR A/A 227 (48.71%)A/C 109 (23.39%)C/C 130 (27.90%)

IL13RA2 rs5946040 Intron 5 G/G 262 (56.47%)G/T 102 (21.98%)T/T 100 (21.55%)

rs10482478 30 UTR G/G 266 (57.82%)G/A 97 (21.09%)A/A 97 (21.09%)

STAT6 rs324010 30 UTR G/G 89 (62.02%) w2¼ 3.64; P¼ 0.16G/A 161 (34.55%)A/A 16 (3.43%)

rs324015 Exon 22 C/C 229 (48.52%) w2 ¼ 0.00; P¼ 1C/T 203 (43.01%)T/T 40 (8.47%)

rs324011 Exon 1 C/C 383 (84.92%) w2¼ 2.36; P¼ 0.31C/T 65 (14.41%)T/T 3 (0.67%)

rs324013 Promoter C/C 170 (36.56%) w2¼ 0.39; P¼ 0.82C/T 235 (50.54%)T/T 60 (12.90%)


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relationship between the probability of an individualdeveloping a high level of infection and the maincovariates known to affect infection by schistosomes.Age and gender were included as covariates in separateanalyses for each polymorphism. A trend of associationwas detected for the rs324013 polymorphism (age:Po10–3; gender: Po10–3 and rs324013: P¼ 0.04).

Thus, rs324013 may be associated with an increasedrisk of infection. We investigated this further by studyingthe genotype distribution over the whole population,taking into account the village of origin and the age ofthe subjects (Figures 1a and b). Subjects carrying thers324013C/T genotype had a higher level of infectionthan those with the C/C or T/T genotypes. This effectwas most marked in subjects from Boul (the villagewith the highest levels of infection) (Figure 1a) and insubjects under 20 years (Figure 1b). We carried outseparate linear regression analyses for subjects under 20years from Segue and Boul. This indicated a significantassociation between rs324013C/T genotype and thehighest infection levels in subjects from Boul but notfrom Segue (P¼ 0.005). In this analysis, both covariates

(age and gender) were excluded. No association wasfound for any of the other markers tested by multivariateanalysis.

rs324013 in STAT6 and rs1800925 in IL13 polymorphismshave an additive effect on infection levelsThe rs324013 polymorphism is the second polymor-phism to be associated with infection levels. Indeed, wepreviously demonstrated that subjects from the samepopulation who carry the rs1800925C/C genotype hadhigher levels of infection than non-carriers. We per-formed a multivariate analysis of infection levels on thetotal population of both Segue and Boul to furthercharacterize the involvement of these two SNPs in thecontrol of the infection levels, with gender and the twoassociated polymorphisms as covariates. We showedthat gender (Po10–3), the rs324013 polymorphism (P¼0.017) and the rs1800925 polymorphism (P¼ 0.027) wereassociated with infection levels. Subjects under 20 years,carrying the rs1800925C/C genotype, had higher infec-tion levels than subjects with other genotypes for thispolymorphism, regardless of rs324013C/T genotype

Table 2 Continued

Gene SNPs Location inthe gene

Genotype Frequency in thestudy population

Hardy–Weinbergequilibrium

test w2 (P-value)a

GATA3 rs444762 Intron 3 C/C 212 (44.17%) w2¼ 0.07; P¼ 0.97C/A 222 (46.25%)A/A 46 (9.58%)

rs406103 Intron 5 C/T 239 (50.00%) w2¼ 0.90; P¼ 0.64C/T 188 (39.33%)T/T 51 (10.67%)

rs477461 30-UTR A/A 302 (63.71%) w2¼ 0.42; P¼ 0.81A/G 151 (31.85%)G/G 21 (4.43%)

Abbreviations: IL, interleukin; SNP, single-nucleotide polymorphism.The distribution of parental genotype for each polymorphism did not deviate from the Hardy–Weinberg equilibrium.aThe frequencies of the IL13RA1 and IL13RA2 polymorphisms were not calculated for parents of the trios, because these markers are locatedon the X chromosome.

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4-12 years old

13-21 years old

22-45 years old

>45 years old

n = 33 24 157 63 133 28 19 17 15 9 94 66 43 31 57 52 38 33n =

Figure 1 rs324013 on STAT6 is associated with high infection levels. (a) Infection levels were quantified by counting eggs in urine. rs324013genotyping of all the subjects from Segue and Boul was performed by TaqMan assay. The mean egg excretion of subjects is shown for subjectsfrom Segue and Boul for each rs324013 genotype. (b) Infection levels for subjects of different age groups, for each rs324013 genotype.


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Genes and Immunity

(Figure 2). This effect was also visible when adjusting forgender or the village of origin (Figure 2). This effect wasmore marked when we limited the analysis to male orBoul inhabitants (Figure 2). Similarly, subjects carryingthe rs324013C/T genotype had higher levels of infectionthan subjects with C/C or T/T variants for thispolymorphism, regardless of rs1800925C/T genotype.

The additive effect of these two markers is illustratedby subjects in Boul with the genotypes rs1800925C/Cand rs324013C/T, exhibiting higher levels of infection(mean¼ 582 eggs per 20 ml) than subjects from thesame population with rs1800925T/T and rs324013C/Cor T/T genotypes (mean¼ 85 eggs per 20 ml). A linearregression analysis confirmed the association of thesetwo genotype combinations with infection levels(P¼ 0.011).

The rs324013 and rs1800925 polymorphisms are not stronglycorrelated with other surrounding SNPsThe rs324013 and rs1800925 polymorphisms may affectinfection levels directly, or they may be correlated withthe causative polymorphisms. The completion of thehuman genome sequence has provided detailed geneticand physical maps of the whole human genome23,24 andan abundance of genetic markers25 (http://www.ensembl.org or http://www.hapmap.org). The SNP frequenciesdescribed in our population (Table 2) are similar to thefrequencies described for the Yoruba reference population(Nigeria). Therefore, the information available on this

reference population was used to select the SNPs locatedwithin the two critical regions and with a minor allelefrequency greater than 20%.

In the 5q31 region, surrounding the IL13 gene, 34 SNPswere genotyped on 87 unrelated subjects (selectedrandomly). Fourteen polymorphisms were genotypedby restriction enzyme analysis (Table 3) and all the otherSNPs were genotyped by TaqMan assay as described inMaterials and methods. The blocks were defined basedon r2 values (Figure 3). The darkest colours indicate thestrongest correlations. The marker rs1800925 located inthe promoter of the gene is not highly correlated with theother tested markers (0or2o0.22). In the 12q13 region,surrounding the STAT6 gene, 14 markers were geno-typed in the same subjects. Five polymorphisms weregenotyped by restriction enzyme analysis (Table 3) andall the other SNPs were genotyped by TaqMan assay. TheSNP rs324013 located in the promoter of the genewas not strongly correlated with any tested markers(0.03or2o0.24; Figure 4).

rs324013 and rs1800925 polymorphisms modify the binding ofnuclear factors to the promoters of STAT6 and IL13 genesSequence changes can influence gene expression throughthe creation or alteration of DNA-binding sites fortranscription factors. The transcription factor databaseTRANSFAC26 was used to analyse in silico thesepolymorphic loci for the potential differences in se-quence similarity to known transcription factor-binding

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C/C C/T T/T

rs324013C/Trs324013C/C or T/T




n = 39 38 71 63 24 26

n =n = 19 25 40 27 10 12 20 13 31 36 14 14

n = 13 9 17 12 2 6 n = 26 29 51 50 22 19

Figure 2 rs1800925 and rs324013 polymorphisms had additive effects on the regulation of infections levels. The association tests indicate thatthe genotypes rs1800925C/C and rs324013C/T are associated with high infection levels (P¼ 0.011). This figure summarizes the distribution ofinfection levels according to these two polymorphisms. This analysis is repeated taking in consideration the gender and the village of origin.


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sites. The threshold score was set at 85% for this analysis(similarity matrix).

In silico analysis of the IL13 gene polymorphismindicated that the C-T transition, at position �1055(rs1800925) from the transcription start codon, affected

the binding of the cap factor to a site that encompassesthe mutation (Figure 5a). In silico analysis of the STAT6polymorphism indicated that the two alleles havedifferent putative binding properties. The rs324013Callele seems to be able to bind to ATF and polyA

Table 3 Markers genotyped by restriction enzyme analysis to determine the blocks of correlation in the 5q31 and 12q13 regions

Region SNPs Positiona Primer sequences(forward/reverse)

PCR productsize

Enzyme Genotype

5q31 rs7737470 132005448 50-CATCAGTGCAGTGGAAGCAC-30 208 bp SspI T/T genotype: 208 bp50-AACGGGTCTTCAGCTTCTTG-30 A/A genotype: 119+89 bp

5q31 rs2069750 132024496 50-GCCAAGGGTTCAGAGACTCA-30 198 bp BsrBI G/G genotype: 156+ 42 bp50-TACTCGTTGGCTGAGAGCTG-30 C/C genotype: 142+42+14 bp

5q31 rs2243206 132028964 50-ATATAGCCGGGGGCTCAG-30 192 bp DdeI C/C genotype: 104+74+14 bp50-TTAGTGGACCCCCAGCAC-30 T/T genotype: 104+44+30+14 bp

5q31 rs2243226 132031509 50-GTATTCCCTCGCCCCTGT-30 245 bp FokI A/A genotype: 111+75+59 bp50-GACCCAGCCAGAGCATTG-30 G/G genotype: 186+59 bp

5q31 rs2243246 132036307 50-CCTGGGTGACAGAGCAAGAT-30 162 bp MseI T/T genotype: 162 bp50-ACCTCCCAGTAAGGGCTCAT-30 C/C genotype: 96+66 bp

5q31 rs2243250 132037498 50-GTAAGGACCTTATGGACCTGC-30 191 bp BsmFI C/C genotype: 115+39+37 bp50-CATCTTGGAAACTGTCCTGTC-30 T/T genotype: 154+37 bp

5q31 rs2243251 132037686 50-CTCATTTTCCCTCGGTTTCA-30 310 bp NheI A/A genotype: 167 + 143 bp50-TGAGAGCATCACCAGAACATC-30 G/G genotype: 310 bp

5q31 rs11479198 132039035 50-GGAGTGAAGCCCTGTGTGTT-30 260 bp HaeIII �/�genotype: 200+60 bp50-GAGGTGGAGGCTGCTATGAG-30 C/C genotype: 166+60+35 bp

5q31 rs2243255 132039636 50-AAGTCCATCCAACCGAGATG-30 207 bp BfmI G/G genotype: 207 bp50-AGTGGCAGAGCTGGGTCTAA-30 A/A genotype: 133+74 bp

5q31 rs2243261 132040705 50-GCTGGCTGATGAAGGGTTT-30 198 bp MseI G/G genotype: 198 bp50-CGATTGGCACTGCATTCAT-30 T/T genotype: 124+74 bp

5q31 rs2243268 132041862 50-GATAGGGGCCTGACAAATGA-30 232 bp HinIII A/A genotype:167+65 bp50-ATCCCACCAGCCAGAGGTA-30 C/C genotype: 232 bp

5q31 rs2243274 132042731 50-TCTGATCTCCCAGGACACC-30 157 bp BsuRI G/G genotype: 81+50+20+12 bp50-CACATCCTTTGGCCTGAACT-30 A/A genotype: 131+20+12 bp

5q31 rs2243283 132044492 50-GTGGACAGAATGGGAGCAGT-30 187 bp HpyCh4IV C/C genotype: 117+70 bp50-CCGCCAGTCTTTCATCTCTC-30 G/G genotype: 187 bp

5q31 rs2243290 132046068 50-CAGGTGACAAGTGCCACAGT-30 163 bp PdmI C/C genotype: 83+80 bp50-TGATCGTCTTTAGCCTTTCCAAGAAGTTTTCCA-30

A/A genotype: 163 bp

12q13 rs324019 55772914 50-AGGGCCTCCCAGTTCTTC-30 212 bp MspI T/T genotype: 212 bp50-CTCCTCCCATCCATGTCG-30 C/C genotype: 132+80 bp

12q13 rs6581128 55869939 50-ACGGAGCTGGCTTTTGTG-30 187 bp PpuMI C/C genotype: 128+58 bp50-CCCTGGGTGTGACAGAGTG-30 A/A genotype: 187 bp

12q13 rs1800154 55875926 50-AGGGCATCAGCCTCACAG-30 203 bp BfuAI C/C genotype: 150+53 bp50-CAGAGGGGAGCCAGGTCT-30 T/T genotype: 203 bp

12q13 rs1800168 55878824 50-CCCGCGAGGACTACATTG-30 206 bp AciI C/C genotype: 155+51+2 bp50-TGCTCTGAACCCCACACC-30 T/T genotype: 206+2 bp

12q13 rs7304504 55892653 50-GGCCCTGAGGACGAGATAG-30 190 bp HphI A/A genotype: 190 bp50-GGCTTGCTCGCTCACATA-30 G/G genotype: 96+94 bp

Abbreviations: IL, interleukin; SNP, single-nucleotide polymorphism.aFor each marker the position according to the coordinate system is indicated.


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rs20

4070

4rs

2040

703

rs77

3747

0rs

1800

925

rs20

6974

3

rs34

2556

86rs

2066

960

rs12

9568

6

rs84

8

rs20

6975

0

rs20

6975

3

rs22

4320

6

rs22

4321

9

rs22

4322

6

rs22

4324

6

rs22

4325

0

rs20

7087

4rs

2243

251

rs73

4244

rs11

4791

98

rs22

4325

5

rs22

4326

1

rs22

4326

6

rs22

4326

7

rs22

4326

8

rs22

4327

0

rs22

4327

4

IL4r

epea

trs

2243

283

rs22

4328

8

rs22

4328

9

rs22

4329

0rs

2243

291

rs11

2421

23

10 kb31LI05DAR IL4 KIF3A

Figure 3 Description of the correlation blocks around the IL13 gene. Thirty four markers, with a minor allele frequency greater than 20%,were genotyped on 87 unrelated subjects (selected randomly). Fourteen markers were genotyped by restriction analysis (Table 3). All theother SNPs were genotyped by TaqMan assay as described in Materials and methods. r2 values and linkage disequilibrium (LD) plot wereobtained with Haploview software (http://www.broad.mit.edu/mpg/haploview/index.php).81 The reported values correspond to ther2 values� 100. The darker colours indicate stronger correlations.

10kb

STAT6 LRP1NAB2

rs32

4019

rs32

4015

rs32

4011

rs32

4013

rs11

1721

13

rs47

5927

7

1183

7145

rs18

0018

1

rs65

8112

8

rs18

0015

4

rs18

0016

8

rs18

0015

9

rs73

0450

4

rs32

4010

Figure 4 Description of the correlation blocks around the STAT6 gene. (a) Thirteen markers, with a minor allele frequency greater than20%, were genotyped on 87 unrelated subjects. Five markers were genotyped by restriction analysis (Table 3). All the other SNPs weregenotyped by TaqMan assay as described in Materials and methods. r2 values and linkage disequilibrium plot were defined as indicatedin Figure 3.


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transcription factors, whereas the rs324013T allele maybind to the NIT2, GATA-2 and GATA-1 transcriptionfactors (Figure 5b).

We performed electrophoretic mobility shift assay,using nuclear extracts from phytohaemagglutinin(PHA)-stimulated peripheral blood mononuclear cells(PBMCs) or from purified CD4þ cells, to determinewhether DNA–protein interactions were altered by thepolymorphisms at either site. The rs1800925T allele fromPHA-stimulated PBMC nuclear extracts forms threeDNA–protein complexes (complexes c1–c3; Figure 5c).The complex c1 is common to both alleles, but lessfrequent on the rs1800925C allele. In nuclear extractsfrom purified CD4þ cells, c1 was detected with the sameintensity level in both alleles (Figure 5d). Analysis of thers324013 polymorphism, using PHA-stimulated PBMCnuclear extract, revealed two DNA–protein complexes(c4 and c5; Figure 5e). Only c5 is common to both alleles.No differential binding between the two alleles wasdetected in nuclear extract from purified CD4þ cells(data not shown).

Discussion

The aim of this study was to investigate the role of theTh2 pathway genes in controlling S. haematobium infec-tion levels in a population from an endemic area. Our

data suggest that the STAT6 gene is essential forimmunity against Schistosome. In particular, we foundthat rs324013 polymorphism is associated with protec-tion against infection in a multivariate analysis.

This SNP was associated with high infection levels inseveral multivariate analyses; thus, the STAT6 gene mayplay a major role in controlling this. The fact that theP-values remain modest after correction is explained by(1) the multigenic nature of processes involved incontrolling infection levels and the presence of additiveeffects between the susceptibility genes; (2) the size ofour study population. However, the Segue/Boul popula-tion is homogenous (only one ethnicity); extending theanalysis to neighbouring villages will introduce someheterogeneity that may mask the association.

STAT6 is the second gene (IL13 being the first) to beassociated with the control of infection levels in S.haematobium infection. We confirmed the presence ofan additive effect between the STAT6 and IL13 poly-morphisms associated with infection levels, in a multi-variate analysis. These two SNPs are not highlycorrelated with any other polymorphism; thus, they playa crucial role in controlling infection levels.

Moreover, we performed functional assays thatshowed that these polymorphisms affect the binding oftranscriptional factors in the promoter regions of the IL13and STAT6 genes. Electrophoretic mobility shift assays ofthe STAT6 promoter, in nuclear extracts from activated

CD4+ Cell’s N.E.rs1800925Crs1800925T

-+-

--+

++-

+-+

PBMC’s N.E.rs324013Crs324013T

-+-

--+

++-

+-+

PBMC’s N.E.rs1800925Crs1800925T

-+-

++-

--+

+-+

c1

c2c3

c1 c4

c5

Figure 5 Electrophoretic mobility shift assay for rs1800925 and rs324013 polymorphisms. The probability of these polymorphisms creatingor altering DNA–protein interaction was determined by in silico analysis (http://www.cbrc.jp/research/db/TFSEARCH.html).26 An 85%threshold score (similarity matrix) was used. For each polymorphism (a, rs1800925; b, rs324013) putative bindings are described. Thesimilarity matrix score is indicated in brackets. Electrophoretic mobility shift assays were performed in vitro as described in Materials andmethods (c and d, rs1800925; e, rs324013). Two different nuclear extracts were used (from PHA-stimulated PBMCs (c, e) or from purifiedCD4þ cells (d)).


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PBMCs, detected differences in DNA–protein interactionproperties between the two tested alleles. This was notdetected in nuclear extracts from CD4þ purified cells,suggesting that the stimulation is necessary for detectionof these differences between the two alleles.

The differential binding properties of the promotercarrying the rs1800925T variant are consistent withrecent data described by Cameron et al.27 Thers1800925T allele is associated with the creation of abinding site for YY1 that overlaps a STAT6 motif.27 TheSTAT6 intracellular mediator has been shown to beessential for polarization of the immune response,leading to high levels of interleukin (IL)-4 and IL-13expression. However, some in vitro studies have shownthat an excess of STAT6 production may inhibit IL-4production.28–30 So, STAT6 may be part of a complexfeedback circuit, acting as an essential inducer of the Th2immune response or as an inhibitor to limit IL-13expression in fully differentiated Th2 cells. In this way,Cameron et al.27 suggested that the creation of a YY1 sitecounteracts STAT6-dependent promoter inhibition. How-ever, the molecular interactions underlying these effectsremain unknown.

No associations were found for polymorphisms in theIL4 gene using multivariate analysis. Moreover, no IL4markers were strongly correlated with the rs1800925polymorphism (r2 values range from 0.05 to 0.22). Thefact that we did not detect an association does notexclude the involvement of this gene in the studiedphenotype. The size of the population may have maskeda potential effect of IL4 polymorphisms in this study.This lack of association may be also explained by the factthat we are studying a homogenous Dogon population.Recently, in Mali, Vafa et al.31 described an associationbetween the IL4-590T allele and Plasmodium falciparumprevalence within the Fulani but not in the Dogon tribe.

In this study, we have shown that IL13 and STAT6polymorphisms are associated to the control ofS. haematobium infection levels. Some polymorphismslocated in the same genes are also associated with thecontrol of infection levels in the endemic area for Ascarislumbricoides, as previously shown in a population fromCameroon (Eboumbou et al., personal communication)and in China.22 These results suggest that these genesplay crucial roles in controlling helminth infection.

Asthma, atopy and allergies are generic terms thatcover a range of aberrant responses to environmentaltargets for which there is a strong genetic basis. Severalpolymorphisms in IL13,32–47 IL4,37,45,48–50 IL4RA,40,51–64

STAT665–72 and GATA370 were associated with asthma,atopy and allergies, indicating that all components of thetype 2 cytokine response pathway involved in suscept-ibility to these disorders.

Our findings suggest that helminth infections influ-ence the allergic response. This is likely because Lynchet al.73 showed that although subjects infected withAscaris lumbricoides had a lower frequency of positiveskin prick test responses to house dust mite allergens,treatment of this parasitic infection increased thefrequency of positive skin prick test responses in theseindividuals. Similarly, there is an inverse associationbetween positive skin prick test responses and theparasitic load of S. mansoni, suggesting that this parasitecould modulate the allergen reactivity in atopic sub-jects.74 Furthermore, a number of studies have demon-

strated negative associations between A lumbricoides,S. mansoni or S. haematobium infection and skin pricktest responses in patients.73–75 Improvements insanitation and the reduction of childhood infectionsthrough vaccinations in developed countries couldshift the balance of the immune response toward atype 2 cytokine response generally associated withallergies.76,77

Materials and methods

Study area—study populationThis study was carried out in the populations of twoDogon villages (Boul and Segue) in the district ofBankass, 200 km from Mopti in Mali. All study subjectswere Dogon; Boul and Segue were built on a hillside andat the top of the hill, respectively. The study populationwas described previously.20,78 All subjects were over 4years of age (693from Segue and 148 from Boul). Thosewho refused to participate or were travelling (2% of thestudy population) were excluded. All subjects includedin this study signed a consent form.

Parasitological methodsS. haematobium infections were quantified by countingeggs in urine.79 Individual infection levels are the meanegg counts in 3–7 samples. Ten per cent of the filters wererandomly selected and re-counted by another micro-scopist. No S. mansoni eggs were found in faeces samplestaken on two different days from 200 young subjects.

Allelic discrimination by PCR with specific TaqMan probesAliquots of 5–15 ml of blood were collected on sodiumcitrate and kept at �20 1C. DNA was extracted using thestandard salting out method.80 Allelic discrimination wasassessed using TaqMan probe assays (Applied Biosys-tems, Lafayette, IN, USA). Each reaction contained12.5 ng of genomic DNA, TaqMan Universal PCR MasterMix (Applied Biosystems), 900 nM of each primer and200 nM of each fluorescently labelled hybridizationprobe in a total volume of 5 ml. reverse transcription-PCR was conducted in an ABI Prism Sequence DetectionSystem 7900 (Applied Biosystems) using the followingconditions: 50 1C for 2 min, 95 1C for 10 min and 40 cyclesof amplification (95 1C denaturation for 15 s, 60 1Cannealing/extension for 1 min).

Allelic discrimination with restriction enzyme analysisPolymerase chain reaction amplifications were carriedout on a robocycler gradient 96 (Stratagene, La Jolla, CA,USA), according to standard protocol.20 Polymorphismswere genotyped by restriction enzyme analysis. Briefly,PCR products specific to each polymorphism wereobtained using the primers described in Table 3. PCRproducts were digested by restriction enzyme understandard conditions described by the enzyme manufac-turers (Euromedex, Mundolsheim, France or NewEngland Biolabs, Beverly, MA, USA). Each digestionwas resolved on either agarose or acrylamide gels,stained with ethidium bromide and visualized by UV.

Nuclear extract preparationNuclear extracts were prepared from PHA-stimulatedhuman PBMCs or purified CD4þ cells. PBMCs werestimulated with 5 mg ml�1 PHA during 12 h. CD4þ cells


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were purified using the EasySep human CD4 selectionkit according to manufacturer’s instructions (StemCellsTechnology, Grenoble, France). Nuclear extracts wereprepared with the nuclear and cytoplasmic extractionreagents from Pierce (NE-PER; Pierce, Rockford, IL,USA). Nuclear protein concentrations were quantifiedwith the bicinchoninic acid protein assay reagent (Pierce,Rockford, IL, USA).

Electrophoretic mobility shift assayComplementary single-stranded oligonucleotides werecommercially synthetized to span approximately 10 bpon either side of the variant nucleotide, as follows:rs1800925: 50 biotin CTTCTAGGAAAA(C/T)GAGGGAAGAGCAGGAA 30

rs324013: 50 biotin CAGAGAGGGAGACGA(C/T)ACAGTTGAAGAGCAGAG 30

Complementary strands were annealed by placingreactions (oligonucleotide and DNA sample) in a boilingwater bath for 10 min and allowing to cool to roomtemperature.

Binding reactions were set up with LightShift Chemi-luminescent EMSA Kit (Pierce, Rockford, IL, USA).Aliquots of 20 fmol of complementary DNA wereincubated at room temperature for 20 min with 4 mgof nuclear extract in 10 mM Tris, 50 mM KCl, 1 mM DTT,2.5% glycerol, 5 mM MgCl2, 50 ngml�1 poly d(I-C), 0.05%NP-40, pH 7.5. Then reactions were loaded onto an 8%non-denaturing polyacrylamide gel and run for 150 minat 110 V. Free DNA and DNA–protein complexes weretransferred to nylon Nþ membrane by capillary action.Binding was detected according to manufacturer0sinstructions (Pierce, Rockford, IL, USA).

Statistical analysisLinkage disequilibrium analysis was performed on thegenepop web site (http://wbiomed.curtin.edu.au/gene-pop/index.html). Multivariate logistic regression wasused to analyse the relationship between the probabilityof an individual developing a high level of infectionand the main covariates known to affect infection byschistosomes. The statistical SPSS software (version 10.0)was used for this analysis. Statistical data presented inthis manuscript were not corrected for multiple testing.However, when we do apply these corrections, the mainassociations remain significant.

Acknowledgements

This work received financial assistance from the InstitutNational de la Sante et de la Recherche Medicale, theWorld Health Organization (ID096546), the EuropeanEconomic Community (TS3 CT940296, IC18CT970212),the Scientific and Technical Cooperation with Develop-ing Countries (IC18CT980373), the French Ministerede la Recherche et des Techniques (PRFMMIP), theConseil General Provence Alpes Cote d’Azur and theConseil Regional Provence Alpes Cote d’Azur. HHreceived a PhD fellowship from the French Embassy inChina.

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Genes and Immunity

a stat6 gene polymorphism is associated with high infection levels in urinary schistosomiasis

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