and Immunity (2007) 8, 79–83 & 2007 Nature Publishing Group All rights reserved 1466-4879/07 $30.00 www.nature.com/gene

SHORT COMMUNICATION Identification of a novel G245R polymorphism in the IL-2 receptor b membrane proximal domain associated with human visceral leishmaniasis

B Bucheton1,2, L Argiro1,3, C Chevillard1,3, S Marquet1,3, MM Kheir4, A Mergani5, SH El-Safi4 and AJ Dessein1,3 1INSERM U399 ‘Immunologie et Ge´ne´tique des Maladies Parasitaires’, Faculte´ de Me´decine La Timone, Marseille, France; 2UMR CNRS/IRD 2724 ‘Ge´ne´tique et Evolution des Maladies Infectieuses (GEMI)’, Montpellier, France; 3Laboratorie de Parasitologie Mycologie, Faculte´ de Me´dicine La Timone, Universite´ de la Me´diterrane´e, Marseille, France; 4Faculty of Medicine, Department of Microbiology and Parasitology, University of Khartoum, Khartoum, Sudan and 5Institute of Nuclear Medicine Molecular Biology and Oncology, University of Gezira, Wad Medani, Sudan

Binding of the -2 (IL-2) to the IL-2 receptor (IL-2R) triggers a series of intracellular events culminating in lymphocyte proliferation and differentiation. We report here the identification of a novel G245R polymorphism in the membrane proximal domain of the IL-2 receptor b chain (IL-2Rb). Present at a frequency of 7.2%, the IL-2-Rb G245R was identified in a population of Eastern Sudan exposed to a severe outbreak of visceral leishmaniasis (VL), a disease associated with a marked depression of T-cell antigen-specific responses. The location of the G245R polymorphism next to the box1/box2 proximal receptor homology segment and suggestive genetic association with the development of disease (P ¼ 0.043), suggest that it may affect (JAK) association and impair growth signal transduction. However, additional genetic association with a synonymous single nucleotide polymorphism (IL2RB þ 8777T) suggests that other variations of IL2RB or nearby genes participate in the highly significant linkage with VL at 22q12 previously reported for this population. Genes and Immunity (2007) 8, 79–83. doi:10.1038/sj..6364355; published online 16 November 2006

Keywords: IL-2 receptor; IL2RB; Leishmaniasis; genetic association; human

Introduction tion. Despite an absolute requirement for the g-chain in signalling, the majority of known signalling pathways The Interleukin-2 (IL-2)/IL-2 receptor (IL-2R) system physically link to the receptor via IL-2Rb, generally plays a central role in the regulation of the magnitude through phosphorylated cytoplasmic residues.7 Stimula- and duration of the T-cell immune response following tion of lymphocytes with IL-2 results in the rapid antigen encounter.1,2 Dysregulated IL-2 signalling has activation of the receptor-associated tyrosine kinases been postulated to play a pathogenic role in a number of JAK1 and JAK3.8,9 Activated JAKs are critical for T-cell-mediated diseases leading to the development of inducing tyrosine phosphorylation of downstream sub- therapeutic strategies aimed at modulating IL-2 signal strates including signal transducer and activator of strength: IL-2 itself is infused in patients with cancer or transcription 5, which then translocates to the nucleus AIDS to enhance T-cell numbers and function;3,4 con- to regulate target gene transcription and cellular pro- versely, anticytokine agents that block IL-2 and/or its liferation.10 receptor are used to inhibit T-cell responses in trans- Leishmania species are obligate intracellular protozoan planted patients5 or rheumatoid arthritis.6 IL-2 mediates parasites of macrophages. Parasitism of the human host its complex biological functions by binding to its specific by Leishmania is asymptomatic in most cases, whereas, receptor consisting of three subunits: IL-2Ra, IL-2Rb and depending on host–parasite complex interactions, some IL-2Rg. The highly inducible a-chain has a very short individuals develop diseases of diverse severity. Leish- cytoplasmic domain and functions to increase the affinity maniasis is well known as a T-cell-mediated disease. for IL-2. In contrast, IL-2R b- and g-chain have longer Studies in the mice experimental models have demon- cytoplasmic domains that participate in signal transduc- strated that a Th1 response is associated with the control of infection by Leishmania major, whereas a Th2 response is associated with disease progression.11 However, in Correspondence: Dr B Bucheton, UMR CNRS/IRD 2724, ‘Ge´ne´tique human visceral leishmaniasis (VL) caused by parasites of et Evolution des Maladies Infectieuses (GEMI)’, Centre IRD de the Leishmania donovani complex, such a dichotomy of the Montpellier, 911 Av Agropolis, 34 394 Montpellier Cedex 5, France. E-mail: [email protected] T-cell-specific response is less clear. The acute phase of Received 15 August 2006; revised 9 October 2006; accepted 9 disease is rather associated with a marked depression October 2006; published online 16 November 2006 of T-cell responses, which has been characterized by IL2RB and human visceral leishmaniasis B Bucheton et al 80 impaired cell proliferation12 and absence of IL-2 pro- subjects. Five were VL-affected children chosen in the duction by lymphocytes on in vitro stimulation with families with highest LOD score at 22q12, and two were Leishmania antigens.13 Noteworthy, this suppression can healthy controls, whom although exposed to Leishmania be reversed in vitro, both in mice14 and human,15 by the infection, did not develop the disease throughout the addition of IL-2, thus suggesting that the T-cell anergic outbreak. In all, 21 single nucleotide polymorphisms state is in part mediated by a defect in IL-2/IL-2R (SNPs) were identified (Figure 1). Seven SNPs are newly signalling.16 Interestingly, the IL2RB gene coding the IL- described polymorphisms of IL2RB among which two 2Rb-chain is located on 22q12 in a region are non-synonymous SNPs (L10V and G245R). Of the 21 for which we found a highly significant genetic linkage identified SNPs, 14 are located in introns; three in the 30 by a genome-wide linkage study of VL susceptibility in UTR and four in the coding sequence of the gene. multicase families of Eastern Sudan17 affected by a severe Interestingly, no SNPs were identified in the 50 and exon outbreak of VL (1996–2000).18,19 This prompts us to 1 regulatory sequence of the gene. One coding sequence investigate whether genetic polymorphisms of the IL2RB SNP was synonymous ( þ 8777 C4T, exon 8) and three gene were associated with resistance/susceptibility to were non-synonymous SNPs. The þ 28 G4C SNP in VL in this population. We report here the identification exon 2 encodes a L10V amino-acid change in the signal of a new sequence variant G245R in the cytoplasmic peptide of the ; the þ 1705 C4T SNP in exon 4 b-chain proximal membrane domain that displayed encodes a S57F polymorphism in the extracellular suggestive genetic association with VL. The potential domain of the protein and the þ 8838 G4A SNP in functional role of this polymorphism in mediating signal exon 8 encodes a non-conservative amino-acid change transduction and its relevance in controlling the devel- G245R introducing a positive charge in the membrane- opment of VL are discussed. proximal region (prox domain) of the cytoplasmic tail. Interestingly, the IL-2-Rb prox domain plays a critical role in the induction of cellular proliferation by mediat- Results and discussion ing JAK1 and JAK3 interaction with IL-2R. In vitro mutational analysis of the prox domain20,21 has enabled Screening of IL2RB variants the identification of several discontinuous amino-acid We analysed global genetic variation in IL2RB by direct residues and protein segments critical for efficient signal re-sequencing of the 50 promoter region, exons and transduction. Noteworthy, the G245R lies just next to intron–exon boundaries and 30 UTR of the gene for seven the box 1/box 2 proximal homology

a EX1 EX2 EX3 EX4 EX5 EX6 EX7 EX8 EX9 EX10

+1 5’ UTR 3’ UTR (rs228941) (rs3218329) (rs228958) (rs228957) (rs3218319) (rs3218327) (rs3218291) (rs3218305) (rs3218306) (rs32183318) (rs228970) (rs2228142) +8924 G>A +5143 T>C +6427 G>C +6649 G>A +8838 G>A +16492 G>C +16521 A>C +112 G>A +500 A>G+500 +91 G>A (rs32182273) +6418 C > G +8692 C > G +8777 C > T +11637 T > C +11644 G > T +28 C > G +1705 C > T +1885 A > G +4885 G>C ( rs228971) +11851 A> C +16169 C> T L10V S57F G245R

extracellular domain cytoplasmic domain SP TM box 1 box 2

b sequenced Fragment +1 Location of the translation start site Figure 1 IL2RB SNPs identified in the study population (Eastern Sudan). Localization of 21 identified SNPs is shown relative to (a): IL2RB transcript; and for non-synonymous SNPs to (b): IL2RB protein sequence and domains (SP ¼ signal peptide; TM ¼ transmembrane domain). Characterization of the IL2RB genetic polymorphisms was carried out by direct re-sequencing of the 50 promoter regions, exons, intron–exon boundaries and 3’ UTR of the gene for seven subjects (sequenced fragments are indicated). Identified SNPs are numbered according to their position relative to the translation start site (position 35870104 in NCBI build 36). For previously published polymorphisms, rs number is given, whereas SNPs newly identified are shown in grey. Apart from the L10V non-synonymous polymorphism in the signal peptide, position of the other amino-acid changes is given relative to the mature protein. Stars indicate polymorphisms typed over the whole familial sample for association analysis.

Genes and Immunity IL2RB and human visceral leishmaniasis B Bucheton et al 81 segment found in a number of type I cytokine receptors Interestingly, the two exon 8 SNPs associated with (Figure 1). Deletions or mutations in box 1 or box 2 VL, although close to each other (61 bp) are in poor regions of IL-2Rb were found to both impair cellular linkage disequilibrium (r2 ¼ 0.045), furthermore, whereas proliferation22 and JAK1 and JAK3 binding.23–25 þ 8777T is associated with protection against VL (P ¼ 0.026), þ 8838A is associated with an increased risk Genetic association with VL of developing VL (P ¼ 0.043). Thus, this argues that these In order to test genetic association with the risk of two IL2RB polymorphisms carry independent effects developing VL, 7 SNPs spanning the IL2RB gene were on VL resistance/susceptibility. The þ 8777C/T poly- genotyped on the whole set of 59 nuclear families (270 morphism is synonymous and has probably no func- individuals) that were used to map a major susceptibility tional effects and is rather in linkage disequilibrium with locus on chromosome 22q12.17 All non-synonymous another causal SNP located in the IL2RB unsequenced variants were included in this analysis as well as four regions or a nearby gene. The fact that the IL2RB þ non-coding SNPs for which the minor allele was at least 8838A/G encodes a non-conservative amino-acid change observed on three of the 14 sequenced . All G245R in a region of the b-chain involved in the early SNPs were in Hardy–Weinberg equilibrium in our popula- events of IL-2 signalling is consistent with a potential tion. Genetic association of these SNPs with VL was functional role of this SNP. The positive charge brought assessed by a Family Based Association Test (FBAT v1.5.5; by R245 could indeed both affect hetero-dimerization of http://www.biostat.harvard.edu/~fbat/default.html), the b- and g-chain upon IL-2 recognition and/or induce which enables testing for genetic association in multicase conformational changes in the IL-2Rb prox domain families in the presence of linkage26 by means of a affecting JAK kinases association/activation. Among transmission disequilibrium test.27 Results are presented other signalling pathways that link to the b-chain in Table 1. Under an additive model, significant ass- cytoplasmic tail, JAK kinases are involved in IL-2- ociations were observed between the two exon 8 SNPs induced cellular proliferation. Thus, the G245R poly- and VL. Associations remained significant when using morphism identified in this Sudanese population could the empirical variance–covariance estimator,28 which provide a clue to explain the anergic state of the CD4 þ is required when sibling marker genotypes are correlated subset during the acute phase of disease.12,13 However, (e.g. when there is linkage and the analysis includes one should note that the IL-2Rb chain is constitutively multiplex families). Similar results were obtained expressed on other cell types than T lymphocytes and for these two SNPs under a dominant model of participates in the activation and differentiation of NK transmission (P ¼ 0.016 and P ¼ 0.041 for IL2RB þ 8777T cells29 and monocytes,30 which indubitably also play a and IL2RB þ 8838A, respectively). Haplotype analysis critical role in the control of Leishmania infections. of the seven genotyped IL2RB SNPs did not show The genetic association observed between the any significant association with VL (data not shown). G245R polymorphism and the risk of VL is borderline

Table 1 Genetic association of IL2RB SNPs with visceral leishmaniasis

SNPa Localization Amino-acid changes Alleles Fq Observed transmissions Expected transmissions P-Valueb Corrected P-Valuec

IL2RB+ 28 Exon 2 Sequence signal G 0.991 8 10.1 L10V C 0.009 6 3.8 0.074 0.18

IL2RB+91 Intron 2 G 0.923 34 33 A 0.077 12 13 0.683

IL2RB+1705 Exon 4 Extracellular C 0.904 75 72.7 S57F T 0.096 23 23.2 0.522

IL2RB+6427 Intron 5 G 0.779 47 47 C 0.221 25 25 1

IL2RB+8777 Exon 8 Synonymous C 0.698 100 91 T 0.302 42 51 0.036 0.026

IL2RB+8838 Exon 8 Prox domain G 0.928 35 41.6 G245R A 0.072 23 16.4 0.02 0.043

IL2RB+11851 Intron 9 C 0.502 99 97.9 A 0.498 77 78 0.83

Genetic association of selected IL2RB SNPs with VL was assessed by mean of a transmission disequilibrium test using the FBAT v5.1.1, which enables to test for association in the presence of linkage by use of an empirical variance-covariance estimator. Results presented were obtained under an additive model of transmission. The family set used for this study has been described elsewhere.17 It was composed of 59 nuclear families comprising 2–5 affected sibs and parents. Primer sequences and genotyping conditions for the SNPs are available upon request. aSNP position relative to the first nucleotide of the IL2RB mRNA (position 35870104 in build 36). bSignificant P-values (o0.05) are in bold. cThe FBAT-e (empirical variance–covariance estimator) function was used to test for genetic association in the presence of linkage.

Genes and Immunity IL2RB and human visceral leishmaniasis B Bucheton et al 82 significant. The sample size used in the frame of this immune function without toxicity. Proc Natl Acad Sci USA study, composed of 59 nuclear families with multiple 1996; 93: 10405–10410. affected children (159) did not have sufficient power to 5 Waldmann TA, O’Shea J. The use of antibodies against the detect genetic association with low frequency SNPs. The IL-2 receptor in transplantation. Curr Opin Immunol 1998; 10: IL2RB þ 8838A allele is only present at 7.2% in our study 507–512. population and only 10 families were informative in the 6 Kozanidou VI, Theocharis AD, Georgiadis A, Voulgari PV, analysis. Association of IL-2-Rb G245R with VL thus Drosos AA, Karamanos NK. Signal transduction by IL-2 requires further confirmation on larger sample size. and its receptors as target in treatment of rheumatoid arthritis. Curr Drug Targets Immune Endocr Metabol Disord 2005; 5: 41–50. Furthermore, given its low frequency and associated 7 Gaffen SL. Signaling domains of the interleukin-2 receptor. P-value, this polymorphism alone is unlikely to explain Cytokine 2001; 14: 63–77. either the high burden of cases that occurred in this 8 Russell SM, Johnston JA, Noguchi M, Kawamura M, Bacon village population during the outbreak (in which more CM, Friedmann M et al. Interaction of IL-2R beta and gamma c than 25% developed VL) or the highly significant LOD chains with Jak1 and Jak3: implications for XSCID and XCID. score observed at 22q12 (LOD ¼ 3.5; P ¼ 0.00003).17 Our Science 1994; 266: 1042–1045. results indeed suggest that other polymorphisms of this 9 Miyazaki T, Kawahara A, Fujii H, Nakagawa Y, Minami Y, genetic region, possibly in linkage disequilibrium with Liu ZJ et al. Functional activation of Jak1 and Jak3 by selective the IL2RB þ 8777C/T synonymous SNP, may also have association with IL-2 receptor subunits. Science 1994; 266: independent effects on the risk of VL. Further work is 1045–1047. 10 Ellery JM, Nicholls PJ. Alternate signalling pathways from required to test if the linkage with VL observed on the interleukin-2 receptor. Cytokine Rev 2002; 13: is due to a major gene or rather to 27–40. genetic variations in multiple genes of the region. 11 Locksley RM, Pingel S, Lacy D, Wakil AE, Bix M, Fowell DJ. Indeed, the locus identified on chromosome 22q12 is Susceptibility to infectious diseases: Leishmania as a paradigm. large (8–10 cM) and encompasses many other genes, J Infect Dis 1999; 179 (Suppl 2): S305–S308. which could be implicated in determining VL suscept- 12 Ho M, Koech DK, Iha DW, Bryceson AD. Immunosuppression ibility. Among those are for example, CSF2RB (granulo- in Kenyan visceral leishmaniasis. Clin Exp Immunol 1983; 51: cyte-macrophage-colony-stimulating factor receptor b- 207–214. chain), NCF4 (p40 PHOX), XBP-1 (X box-binding protein 13 Carvalho EM, Badaro R, Reed SG, Jones TC, Johnson Jr WD. 1) or LIF (leukaemia inhibitory factor). Linkage disequi- Absence of gamma and interleukin-2 production J Clin Invest 76 librium mapping in 22q12 is now required to narrow during active visceral leishmaniasis. 1985; : 2066–2069. down (or split) the susceptibility locus interval in order 14 Eslami Z, Olivier M, Tanner CE. Immunotherapy with IL-2- to choose positional candidate for the analysis of the VL stimulated splenocytes reduces in vitro the level of Leishmania resistance/susceptibility phenotype. donovani infection in peritoneal macrophages. Int J Parasitol In conclusion, we have identified a novel polymorph- 1995; 25: 975–981. ism encoding a non-conservative amino-acid change in 15 Carvalho EM, Bacellar O, Brownell C, Regis T, Coffman RL, the proximal membrane cytoplasmic domain of the IL- Reed SG. Restoration of IFN-gamma production and lympho- 2Rb-chain. Location of the G245R polymorphism in a cyte proliferation in visceral leishmaniasis. J Immunol 1994; protein domain implicated in signal transduction and 152: 5949–5956. genetic association with VL suggest that this polymorph- 16 Barral-Netto M, Barral A, Santos SB, Carvalho EM, Badaro R, ism may have a functional effect on IL-2-mediated Rocha H et al. Soluble IL-2 receptor as an agent of serum- mediated suppression in human visceral leishmaniasis. signalling. Whereas this needs to be confirmed by in J Immunol 1991; 147: 281–284. vitro functional studies, we believe that this polymor- 17 Bucheton B, Abel L, El-Safi S, Kheir MM, Pavek S, Lemainque phism is worth testing in other populations (especially A et al. A major susceptibility locus on chromosome 22q12 those of African descent) affected by VL or other T-cell- plays a critical role in the control of kala-azar. Am J Hum Genet mediated disease. It could also provide an interesting 2003; 73: 1052–1060. marker for the analysis of IL-2 responsiveness in patients 18 Bucheton B, Kheir MM, El-Safi SH, Hammad A, Mergani A, receiving IL-2 immunotherapy. Mary C et al. The interplay between environmental and host factors during an outbreak of visceral leishmaniasis in eastern Sudan. Microbes Infect 2002; 4: 1449–1457. 19 EL-Safi S, Bucheton B, Kheir MM, Musa HA, M EL-O, Acknowledgements Hammad A et al. Epidemiology of visceral leishmaniasis in Atbara River area, eastern Sudan: the outbreak of Barbar El This work was supported by a Grant IC 18 CT 98 0373 Fugara village (1996–1997). Microbes Infect 2002; 4: 1439–1447. (European Commission) by the French Research Ministry 20 Howard OM, Kirken RA, Garcia GG, Hackett RH, Farrar WL. Programme PRFMMIP/Microbiology and by INSERM. Structural domains of interleukin-2 receptor beta critical for signal transduction: kinase association and nuclear complex- formation. Biochem J 1995; 306 (Part 1): 217–224. References 21 Liu KD, Lai SY, Goldsmith MA, Greene WC. Identification of a variable region within the cytoplasmic tail of the IL-2 receptor 1 Lin JX, Leonard WJ. Signaling from the IL-2 receptor to the beta chain that is required for growth signal transduction. nucleus. Cytokine Growth Factor Rev 1997; 8: 313–332. J Biol Chem 1995; 270: 22176–22181. 2 Nelson BH. IL-2, regulatory T cells, and tolerance. J Immunol 22 Goldsmith MA, Xu W, Amaral MC, Kuczek ES, Greene WC. 2004; 172: 3983–3988. The cytoplasmic domain of the interleukin-2 receptor 3 Dutcher J. Current status of interleukin-2 therapy for meta- beta chain contains both unique and functionally redun- static renal cell carcinoma and metastatic melanoma. Oncology dant signal transduction elements. J Biol Chem 1994; 269: (Williston Park) 2002; 16 (11 Suppl 13): 4–10. 14698–14704. 4 Jacobson EL, Pilaro F, Smith KA. Rational interleukin-2 23 Kirken RA, Rui H, Malabarba MG, Howard OM, Kawamura therapy for HIV positive individuals: daily low doses enhance M, O’Shea JJ et al. Activation of JAK3, but not JAK1, is critical

Genes and Immunity IL2RB and human visceral leishmaniasis B Bucheton et al 83 for IL-2-induced proliferation and STAT5 recruitment by a 27 Spielman RS, McGinnis RE, Ewens WJ. Transmission test for COOH-terminal region of the IL-2 receptor beta-chain. linkage disequilibrium: the insulin gene region and insulin- Cytokine 1995; 7: 689–700. dependent diabetes mellitus (IDDM). Am J Hum Genet 1993; 24 Zhu MH, Berry JA, Russell SM, Leonard WJ. Delineation of 52: 506–516. the regions of interleukin-2 (IL-2) receptor beta chain 28 Lake SL, Blacker D, Laird NM. Family-based tests of association important for association of Jak1 and Jak3. Jak1-independent in the presence of linkage. Am J Hum Genet 2000; 67: 1515–1525. functional recruitment of Jak3 to IL-2Rbeta. J Biol Chem 1998; 29 Nishikawa K, Saito S, Morii T, Kato Y, Narita N, Ichijo M et al. 273: 10719–10725. Differential expression of the interleukin-2 receptor beta (p75) 25 Ihle JN, Witthuhn BA, Quelle FW, Yamamoto K, Silvennoinen chain on human peripheral blood natural killer subsets. Int O. Signaling through the hematopoietic cytokine receptors. Immunol 1990; 2: 481–486. Annu Rev Immunol 1995; 13: 369–398. 30 Espinoza-Delgado I, Ortaldo JR, Winkler-Pickett R, Sugamura 26 Laird NM, Horvath S, Xu X. Implementing a unified approach K, Varesio L, Longo DL. Expression and role of p75 to family-based tests of association. Genet Epidemiol 2000; 19 interleukin-2 receptor on human monocytes. J Exp Med 1990; (Suppl 1): S36–S42. 171: 1821–1826.

Genes and Immunity