Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Genes Immun. Author manuscript; available in PMC 2012 Mar 9.
Published in final edited form as:
PMCID: PMC3297968
NIHMSID: NIHMS360954

FLI1 polymorphism affects susceptibility to cutaneous leishmaniasis in Brazil

Abstract

Mapping murine genes controlling cutaneous leishmaniasis (CL) identified Fli1 as a candidate influencing resistance to L. major and enhanced wound healing. We examine FLI1 as a gene controlling CL and mucosal leishmaniasis (ML) caused by L. braziliensis in humans. Intron 1 single nucleotide polymorphisms tagging promoter and enhancer elements were analysed in 168 nuclear families (250 CL; 87 ML cases) and replicated in 157 families (402 CL; 39 ML cases). Robust case-pseudocontrol logistic regression analysis showed association between allele C (odds ratio (OR) 1.65; 95% confidence interval 1.18–2.29; P = 0.003) of FLI1_rs7930515 and CL in the primary sample that was confirmed (OR 1.60; 95% confidence interval 1.10–2.33; P = 0.014) in the replication set (combined P = 1.8 × 10−4). FLI1_rs7930515 is in linkage disequilibrium with the functional GAn microsatellite in the proximal promoter. Haplotype associations extended across the enhancer, which was not polymorphic. ML associated with inverse haplotypes compared with CL. Wound healing is therefore important in CL, providing potential for therapies modulating FLI1.

Keywords: FLI1, cutaneous leishmaniasis, wound healing, genetic susceptibility

Leishmania infection is associated with a broad spectrum of clinical phenotypes and many studies have demonstrated that host genetic factors have a part in determining the outcome of infection (reviewed in refs. 14). L. braziliensis infection causes cutaneous leishmaniasis (CL) with prolonged time to lesion healing. Pro-inflammatory cytokines, including tumour necrosis factor and interferon-γ, and macrophage activation are important in eventual self-healing, but an exaggerated response is associated with mucosal leishmaniasis (ML).5,6 A number of studies711 have now been published that report on the role of polymorphisms at candidate genes (TNFA, SLC11A1, CXCR1, IL6, IL10, MCP1) associated with pro- and anti-inflammatory responses in regulating clinical disease outcome in L. braziliensis infection in humans. These studies have generally been carried out using small sample sizes and without replication in a second sample. Nevertheless, they have been underpinned by functional data911 and/or are supported by previous immunological studies5,6,1214 demonstrating the importance of these pathways in determining disease outcome. Two of these studies8,9 were undertaken using the set of families that we use here as a primary sample set, now strengthened by collection of a new replication sample. Thus far no genome-wide approaches to identify novel genes contributing to clinical outcome in L. braziliensis infection have been reported.

One alternative way to identify genes regulating clinical outcome in humans is via a mouse-to-human approach. This has proven quite successful in identifying genes that regulate visceral leishmaniasis in humans.1 In the mouse, genome-wide linking mapping of genes controlling CL caused by L. major has demonstrated complex genetic control,3,4,1518 among which a particular role for wound healing genes has been proposed.19 Recent fine mapping in the region of chromosome 9 in mice (chromosome 11q24 in humans) has identified Fli1 (Friend leukaemia virus integration 1; FLI1 in humans) as a novel candidate influencing both resistance to L. major and enhanced wound-healing responses.20 We also recently reported8 associations between CL caused by L. braziliensis infection in humans and polymorphisms at CXCR1/SLC11A1, which we interpreted in relation to their roles in regulating polymorphonuclear neutrophils, macrophages and/or dendritic cells in wound-healing responses. Here we report on primary and replication family-based genetic association studies that support a role for polymorphism at FLI1 in determining susceptibility to CL caused by L. braziliensis in Brazil.

To undertake our study, we selected four single nucleotide polymorphisms (SNPs; Table 1) that tagged the first two of four major linkage disequilibrium blocks in the large (>50 kb) intron 1 of the FLI1 gene (Figure 1a). The SNPs selected also tagged the proximal promoter region that contains a functional GAn microsatellite,21 as well as the CpG island that spans the proximal promoter region and the 5′ region of intron 1 (Figure 1a). We focused on this region extending into intron 1 of FLI1 because a previous study22 had demonstrated a functional ETS/ETS/GATA (E-twenty six family of transcription factors;23 GATA family of transcription factors that bind to the sequence GATA24) enhancer element residing in this intron (Figure 1a). FLI1 is itself a member of the ERG sub-family of ETS transcription factors.23 The 4 tag-SNPs (Table 1) had minor allele frequencies >0.1 in the HapMap populations representative of the three major ethnicities (Caucasian, African and Asian) that contribute to this population in Brazil.

Figure 1
Gene structure of FLI1 showing position of SNPs used in this study in relation to linkage disequilibrium (LD) blocks and functional regulatory elements. (a) A diagrammatic representation of gene structure, the location of the GAn repeat and the ETS/ETS/GATA ...
Table 1
Details of the FLI1 intron 1-genotyped SNPs including allele frequencies for Caucasian (CEPH), Asian (HCB) and sub-Saharan (YRI) populations as recorded in Build 37.1 of the NCBI Entrez SNP database32

Initially we genotyped these SNPs in 168 nuclear families (Table 2) used in our previous studies8,9 that contain 250 CL cases and 87 ML cases collected in the area of Corte de Pedra, Bahia, Brazil, where L. braziliensis is endemic. Using robust case-pseudocontrol conditional logistic regression analysis (Table 3), we demonstrated association between the common allele C (odds ratio (OR) 1.64; P = 0.003) at SNP rs7930515 and risk of CL, which was replicated (OR 1.60; P = 0.014) in a second sample (Table 2) of 402 CL cases in 157 nuclear families (combined OR 1.62; P = 1.8 × 10−4; Table 3) collected in the same region of Corte de Pedra. Application of a Bonferroni correction for four independent SNPs provides a significance cutoff of P ≤ 0.013 (that is, P = 0.05/4), which is achieved at rs7930515 in primary and combined samples. Power to detect association with ML disease in our study is limited by sample size. Single point association analysis undertaken in TRANSMIT (http://www-gene.cimr.cam.ac.uk/clayton/software/transmit.txt) (Figure 2) using the combined primary and replication samples was suggestive of association at this SNP for ML. This showed overtransmission of the minor allele A at SNP rs7930515, consistent with the observation that analysis of CL and ML cases together as leishmaniasis per se showed reduced significance compared with analysis of cases with a long history of CL only disease. Haplotype analysis in TRANSMIT (Figure 2 and Supplementary Figure 1) demonstrated (a) that haplotypes controlling CL extended across the remaining tag-SNPs, strongest over the region containing the enhancer element, and (b) that ML was associated with the inverse 2.1.2 ( = A.A.T) rs793515_rs6199521_rs590520 haplotype compared with CL (1.2.1 = C.G.C; Figure 2). Data for one degree of freedom tests for specific haplotype associations presented in Figure 2 are supported by global test statistics (Supplementary Figure 1) in which the total number of haplotypes is taken into account as indicated by the degrees of freedom. Diminishing strength of the CL haplotype associations (Figure 2; Supplementary Figure 1) moving 3′ across the four SNPs concurs with the observation in this Brazilian population that, although D′ between rs7930515 and rs619521 (D′ = 0.62) and between rs590520 and rs531894 (D′ = 0.86) are relatively high, r2 values that take allele frequencies into account are low (0.11 and 0.29, respectively) and below the stringent cutoff (r2 = 0.8) we set in choosing tag SNPs. In summary, the association between FLI1 and CL observed in primary and replication samples appears to map within linkage disequilibrium block 1 (Figure 1b) to the region that is most strongly tagged by SNP rs7930515 and closest to the proximal promoter containing the functional GAn microsatellite repeat, with haplotype associations extending across the region of intron 1 containing the functional ETS/ETS/GATA enhancer element proximal of SNP rs619521. Interestingly, no SNPs are observed in public domain databases within the region of the ETS/ETS/GATA enhancer element (Supplementary Figure 2), attesting to its importance as a highly conserved regulatory element.22 The FLI1 promoter is itself upregulated by ETS factors ETS1, ETS2, FLI1 and ELF1, in combination with GATA factors.25 Recent improved motif identification and analysis of ChIP-Seq data demonstrated that FLI1 activates gene transcription when its binding site is located in close proximity to the gene transcription start site (up to ~150 kb), especially when it contains a microsatellite sequence.26 Hence, there may be strong functional significance to the maintenance of linkage disequilibrium across the proximal promoter and intron 1 of FLI1 containing the GAn microsatellite and the ETS/ETS/GATA enhancer.

Figure 2
Results of the single point (global P-value) and haplotype (1 degree of freedom tests for individual haplotypes) analyses performed in TRANSMIT for combined families from primary and replication samples. TRANSMIT analyses, which use the EM algorithm to ...
Table 2
Characteristics of the primary and replication samples
Table 3
Results of robust CPC analysis for FLI1 tag SNP rs7930515 (see Table 1 and Figure 1) for transmission of alleles CPC analysis for transmission of alleles from heterozygous parents to CL, ML and L. braziliensis per se (CL and ML) individuals in families ...

Here we have demonstrated association (combined OR 1.62; 95% confidence interval 1.26–2.09; P = 1.8 × 10−4) between FLI1 and susceptibility to CL caused by L. braziliensis. This follows on from genetic and functional mapping of the lmr2 gene controlling cutaneous lesions caused by L. major on murine chromosome 9 to the Fli1 gene.20 Resistance to L. major correlated with a wound-healing response that presented in congenic resistant mice as a large population of fibroblasts and an organized and abundant deposition of collagen bundles in the absence of inflammatory cells. Recent studies have shown an association between enhanced type I collagen expression and epigenetic repression of the FLI1 gene.27 In congenic susceptible mice, response to wounding was associated with a larger population of acute inflammatory cells with sparse and disorganized collagen bundles. In a previous study9 we found an association between CL caused by L. braziliensis and a regulatory polymorphism in the promoter of the gene IL6 encoding interleukin 6. Homocysteine-dependent stimulation of interleukin 6 has recently been reported28 to upregulate genes essential for epigenetic DNA methylation (DNA (cytosine-5-)-methyl-transferases (Dnmts) via expression of FLI1. Homocysteine increases the CpG methylation status (and hence represses gene expression) of the CpG-rich proximal promoter of the lysyl oxidase (LOX) gene,28 an extra-cellular copper enzyme that initiates the cross-linking of collagens and elastins. Inhibition of interleukin 6 reverses this repression. Regulation of collagen expression and organization may thus involve epigenetic regulation at both FLI1 and LOX genes, consistent with the presence of the CpG motif across the region of the functional FLI1 promoter elements. This suggests that, although there are many immune-related functions for both interleukin 6 and FLI1 that could account for association with CL caused by L. braziliensis, there may be a direct functional link between these two genes that mediates resistance or susceptibility to infection through the wound-healing response. This, in turn, might provide a novel therapeutic opportunity. For example, the use of imatinib mesylate has been proposed for treatment of systemic sclerosis,29,30 an autoimmune disorder similarly resulting from immune activation, fibrosis development and damage of small blood vessels, in which FLI1 is downregulated through an epigenetic mechanism.30 Imatinib mesylate reverses the expression levels of FLI1. Further work will be required to analyse expression levels of FLI1 in tissue biopsies from L. braziliensis patients to determine its potential as a therapeutic target. Overall our results strengthen the potential role of genes/mechanisms associated with wound healing in CL, and suggest that a broader analysis of pathways involved in wound-healing responses may contribute to a better understanding of the pathogenesis of disease.

Acknowledgments

We acknowledge the support of NIH Grant AI 30639 for the field work in Brazil, and The Wellcome Trust for supporting the laboratory work and statistical analyses carried out in the UK. LC was supported by NIH/FIC 1 D43 TW007127–01 for her period of stay in UK. JO and AM were also supported by NIH/FIC 1 D43 TW007127-01 in Brazil.

Footnotes

Conflict of interest

The authors declare no conflict of interest.

Supplementary Information accompanies the paper on Genes and Immunity website (http://www.nature.com/gene)

Authors contributions: LC carried out the field collection and preparation of the samples, performed the genotyping, and participated in the statistical analysis and interpretation of the data. SEJ and ENM trained LC in the laboratory for genotyping techniques, in database entry and use of the genetic database GenIE in Cambridge, and in genetic statistical analysis methods. SEJ cross-checked statistical analyses and carried out additional statistical tests. LFA, JO, AM and LHG participated in the field collection of data, processing of DNA samples and database entry in Brazil. ML is the doctor responsible for confirmation of the ML cases by performing ENT exams. EL participated in the field work by contacting patients and helping sample collection. ARJ trained the field group, initial selection of cases from the health post, assisted with field collection of data and participated in the design of the study. EMC helped conceive the study, initial selection of cases from the health post and provided the logistical support to make the study possible. JMB participated in the design of the study, conceived the specific hypothesis to be tested, made the final interpretation of the data and prepared the manuscript. All authors read and approved the final manuscript.

References

1. Blackwell JM, Fakiola M, Ibrahim ME, Jamieson SE, Jeronimo SB, Miller EN, et al. Genetics and visceral leishmaniasis: of mice and man. Parasite Immunol. 2009;31:254–266. [PMC free article] [PubMed]
2. El-Safi S, Kheir MM, Bucheton B, Argiro L, Abel L, Dereure J, et al. Genes and environment in susceptibility to visceral leishmaniasis. C R Biol. 2006;329:863–870. [PubMed]
3. Sakthianandeswaren A, Foote SJ, Handman E. The role of host genetics in leishmaniasis. Trends Parasitol. 2009;25:383–391. [PubMed]
4. Lipoldova M, Demant P. Genetic susceptibility to infectious disease: lessons from mouse models of leishmaniasis. Nat Rev Genet. 2006;7:294–305. [PubMed]
5. Castes M, Trujillo D, Rojas ME, Fernandez CT, Araya L, Cabrera M, et al. Serum levels of tumor necrosis factor in patients with American cutaneous leishmaniasis. Biol Res. 1993;26:233–238. [PubMed]
6. Bacellar O, Lessa H, Schriefer A, Machado P, Ribeiro de Jesus A, Dutra WO, et al. Up-regulation of Th1-type responses in mucosal leishmaniasis patients. Infect Immun. 2002;70:6734–6740. [PMC free article] [PubMed]
7. Cabrera M, Shaw M-A, Sharples C, Williams H, Castes M, Convit J, et al. Polymorphism in TNF genes associated with mucocutaneous leishmaniasis. J Exp Med. 1995;182:1259–1264. [PMC free article] [PubMed]
8. Castellucci L, Jamieson SE, Miller EN, Menezes E, Oliveira J, Magalhaes A, et al. CXCR1 and SLC11A1 polymorphisms affect susceptibility to cutaneous leishmaniasis in Brazil: a case-control and family-based study. BMC Med Genet. 2010;11:10. [PMC free article] [PubMed]
9. Castellucci L, Menezes E, Oliveira J, Magalhaes A, Guimaraes LH, Lessa M, et al. IL6 -174 G/C promoter polymorphism influences susceptibility to mucosal but not localized cutaneous leishmaniasis in Brazil. J Infect Dis. 2006;194:519–527. [PubMed]
10. Salhi A, Rodrigues V, Jr, Santoro F, Dessein H, Romano A, Castellano LR, et al. Immunological and genetic evidence for a crucial role of IL-10 in cutaneous lesions in humans infected with Leishmania braziliensis. J Immunol. 2008;180:6139–6148. [PubMed]
11. Ramasawmy R, Menezes E, Magalhaes A, Oliveira J, Castellucci L, Almeida R, et al. The-2518bp promoter polymorphism at CCL2/MCP1 influences susceptibility to mucosal but not localized cutaneous leishmaniasis in Brazil. Infect Genet Evol. 2010;10:607–613. [PMC free article] [PubMed]
12. Lessa HA, Machado P, Lima F, Cruz AA, Bacellar O, Guerreiro J, et al. Successful treatment of refractory mucosal leishmaniasis with pentoxifylline plus antimony. Am J Trop Med Hyg. 2001;65:87–89. [PubMed]
13. D’Oliveira A, Jr, Machado P, Bacellar O, Cheng LH, Almeida RP, Carvalho EM. Evaluation of IFN-gamma and TNF-alpha as immunological markers of clinical outcome in cutaneous leishmaniasis. Rev Soc Bras Med Trop. 2002;35:7–10. [PubMed]
14. Faria DR, Gollob KJ, Barbosa JJ, Schriefer A, Machado PR, Lessa H, et al. Decreased in situ expression of interleukin-10 receptor is correlated with the exacerbated inflammatory and cytotoxic responses observed in mucosal leishmaniasis. Infect Immun. 2005;73:7853–7859. [PMC free article] [PubMed]
15. Beebe AM, Mauze S, Schork NJ, Coffman RL. Serial backcross mapping of multiple loci associated with resistance to Leishmania major in mice. Immunity. 1997;6:551–557. [PubMed]
16. Mock B, Blackwell J, Hilgers J, Potter M, Nacy C. Genetic control of Leishmania major infection in congenic, recombinant inbred and F2 populations of mice. Eur J Immunogenet. 1993;20:335–348. [PubMed]
17. Roberts M, Mock BA, Blackwell JM. Mapping of genes controlling Leishmania major infection in CXS recombinant inbred mice. Eur J Immunogenet. 1993;20:349–362. [PubMed]
18. Havelkova H, Badalova J, Svobodova M, Vojtikova J, Kurey I, Vladimirov V, et al. Genetics of susceptibility to leishmaniasis in mice: four novel loci and functional heterogeneity of gene effects. Genes Immun. 2006;7:220–233. [PubMed]
19. Sakthianandeswaren A, Elso CM, Simpson K, Curtis JM, Kumar B, Speed TP, et al. The wound repair response controls outcome to cutaneous leishmaniasis. Proc Natl Acad Sci USA. 2005;102:15551–15556. [PMC free article] [PubMed]
20. Sakthianandeswaren A, Curtis JM, Elso C, Kumar B, Baldwin TM, Lopaticki S, et al. Fine mapping of Leishmania major susceptibility Locus lmr2 and evidence of a role for Fli1 in disease and wound healing. Infect Immun. 2010;78:2734–2744. [PMC free article] [PubMed]
21. Morris EE, Amria MY, Kistner-Griffin E, Svenson JL, Kamen DL, Gilkeson GS, et al. A GA microsatellite in the Fli1 promoter modulates gene expression and is associated with systemic lupus erythematosus patients without nephritis. Arthritis Res Ther. 2010;12:R212. [PMC free article] [PubMed]
22. Donaldson IJ, Chapman M, Kinston S, Landry JR, Knezevic K, Piltz S, et al. Genome-wide identification of cis-regulatory sequences controlling blood and endothelial development. Hum Mol Genet. 2005;14:595–601. [PubMed]
23. Sharrocks AD. The ETS-domain transcription factor family. Nat Rev Mol Cell Biol. 2001;2:827–837. [PubMed]
24. Ko LJ, Engel JD. DNA-binding specificities of the GATA transcription factor family. Mol Cell Biol. 1993;13:4011–4022. [PMC free article] [PubMed]
25. Svenson JL, Chike-Harris K, Amria MY, Nowling TK. The mouse and human Fli1 genes are similarly regulated by Ets factors in T cells. Genes Immun. 2010;11:161–172. [PMC free article] [PubMed]
26. Boeva V, Surdez D, Guillon N, Tirode F, Fejes AP, Delattre O, et al. De novo motif identification improves the accuracy of predicting transcription factor binding sites in ChIP-Seq data analysis. Nucleic Acids Res. 2010;38:e126. [PMC free article] [PubMed]
27. Wang Y, Fan PS, Kahaleh B. Association between enhanced type I collagen expression and epigenetic repression of the FLI1 gene in scleroderma fibroblasts. Arthritis Rheum. 2006;54:2271–2279. [PubMed]
28. Thaler R, Agsten M, Spitzer S, Paschalis EP, Karlic H, Klaushofer K, et al. Homocysteine suppresses the expression of the collagen cross-linker lysyl oxidase involving IL-6, Fli1 and epigenetic DNA-methylation. J Biol Chem. 2010;286:5578–5588. [PMC free article] [PubMed]
29. Asano Y. Future treatments in systemic sclerosis. J Dermatol. 2010;37:54–70. [PubMed]
30. Asano Y, Bujor AM, Trojanowska M. The impact of Fli1 deficiency on the pathogenesis of systemic sclerosis. J Dermatol Sci. 2010;59:153–162. [PMC free article] [PubMed]
31. Clayton D. A generalization of the transmission/disequilibrium test for uncertain-haplotype transmission. Am J Hum Genet. 1999;65:1170–1177. [PMC free article] [PubMed]
32. NCBI. Entrez SNP. 2010 http://wwwncbinlmnihgov/sites/entrez.
33. Knapp M. A note on power approximations for the transmission disequilibrium test. Am J Hum Genet. 1999;64:1177–1185. [PMC free article] [PubMed]
34. Marsden PD. Mucosal leishmaniasis (‘espundia’ Escomel, 1911) Trans R Soc Trop Med Hyg. 1986;80:859–876. [PubMed]
35. O’Connell JR, Weeks DE. PedCheck: a program for identification of genotype incompatibilities in linkage analysis. Am J Hum Genet. 1998;63:259–266. [PMC free article] [PubMed]
36. Cordell HJ, Barratt BJ, Clayton DG. Case/pseudocontrol analysis in genetic association studies: A unified framework for detection of genotype and haplotype associations, gene-gene and gene-environment interactions, and parent-of-origin effects. Genet Epidemiol. 2004;26:167–185. [PubMed]
PubReader format: click here to try

Formats:

Save items

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Gene
    Gene
    Gene records that cite the current articles. Citations in Gene are added manually by NCBI or imported from outside public resources.
  • GEO Profiles
    GEO Profiles
    Gene Expression Omnibus (GEO) Profiles of molecular abundance data. The current articles are references on the Gene record associated with the GEO profile.
  • HomoloGene
    HomoloGene
    HomoloGene clusters of homologous genes and sequences that cite the current articles. These are references on the Gene and sequence records in the HomoloGene entry.
  • MedGen
    MedGen
    Related information in MedGen
  • PubMed
    PubMed
    PubMed citations for these articles

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...