Genetic Variants of RANTES Are Associated with Serum RANTES Level and Protection for Type 1 Diabetes

ORIGINAL ARTICLE Genetic variants of RANTES are associated with serum RANTES level and protection for type 1 diabetes

A Zhernakova1, BZ Alizadeh1, P Eerligh2, P Hanifi-Moghaddam2,3, NC Schloot3, B Diosdado1, C Wijmenga1, BO Roep2 and BPC Koeleman1,2 1Department of Medical Genetics, University Medical Centre Utrecht, Utrecht, The Netherlands; 2Department of Immunohematology and Blood Transfusion, Leiden University Medical Centre, Leiden, The Netherlands and 3Diabetes Research Institute, Heinrich-Heine University, Dusseldorf, Germany

RANTES (regulated on activation, normal T-cell expressed and secreted) is a T-helper type 1 (Th1) chemokine that promotes T-cell activation and proliferation. RANTES is genetically associated with asthma, sarcoidosis and multiple sclerosis. The concentration of RANTES is increased at inflammation sites in different autoimmune diseases. Type 1 diabetes (T1D) is a Th1-mediated disease with complex genetic predisposition. We tested RANTES as a candidate gene for association with T1D using three single-nucleotide polymorphism (SNP) variants (rs4251719, rs2306630 and rs2107538) to capture haplotype information. The minor alleles of all SNPs were transmitted less frequently to T1D offspring (transmission rates 37.3% (P ¼ 0.002), 38.7% (P ¼ 0.007) and 41.0% (P ¼ 0.01)) and were less frequently present in patients compared to controls (P ¼ 0.009, 0.03 and 0.04, respectively). A similar protective effect was observed for the haplotype carrying three minor alleles (transmission disequilibrium test (TDT): P ¼ 0.003; odds ratio (OR) ¼ 0.55; confidence interval (CI): 0.37–0.83; case/control: P ¼ 0.03; OR ¼ 0.74; CI: 0.55–0.98). Both patients and controls carrying the protective haplotype express significantly lower serum levels of RANTES compared to non-carriers. Subsequently, we tested a cohort of 310 celiac disease patients, but failed to detect association. RANTES SNPs are significantly associated with RANTES serum concentration and development of T1D. The rs4251719*A–rs2306630*A–rs2107538*A haplotype associated with low RANTES production confers protection from T1D. Our data imply that RANTES is associated with T1D both genetically and functionally, and contributes to diabetes- prone Th1 cytokine profile. Genes and Immunity (2006) 7, 544–549. doi:10.1038/sj.gene.6364326; published online 20 July 2006

Keywords: RANTES; CCL5; autoimmune diseases; type 1 diabetes; celiac disease; Th1 cytokine profile

Introduction CCL5). This gene has not yet been investigated in the context of T1D, although it has been shown to be Type 1 diabetes (T1D) is an autoimmune disease associated with other autoimmune diseases, such characterized by a T-cell-mediated progressive destruc- as atopic dermatitis, asthma, sarcoidosis, rheumatoid tion of pancreatic beta cells.1 The genetic predisposition arthritis and multiple sclerosis.5–12 RANTES belongs to to T1D is complex, implying that multiple susceptibility the family of CC chemokines, which are involved in genes with different effects play a role in the develop- immunoregulatory and inflammatory processes owing to ment of the disease. Approximately, 50% of the genetic their ability to recruit, activate and co-stimulate T cells susceptibility to T1D is explained by four established and monocytes.13,14 In addition to the trafficking effect, genetic risk factors, including human leukocyte antigen RANTES, like other CC chemokines, plays an important class II genes, the insulin gene, cytotoxic T-lymphocyte- role in co-stimulation of T-cell proliferation15,16 and associated antigen 4 (CTLA4) and protein tyrosine activation of the T cells localized in the inflammatory phosphatase PTPN22.2–4 These findings indicate that lesion.17 Activation of T cells by RANTES leads to analyzing candidate genes is a fruitful approach in the interleukin-2 (IL-2) receptor expression, cytokine release search for T1D susceptibility genes, but that other genetic and T-cell proliferation.16 Micromolar concentrations of risk factors still need to be identified. RANTES can induce T-cell proliferation in an antigen- An interesting candidate gene is RANTES (‘regulated independent manner that is especially important in on activation, normal T-cell expressed and secreted’, autoimmunity, whereas the increased level of RANTES in the inflammatory sites may lead to the enhanced activation and proliferation of local T cells.17 Recent studies Correspondence: Dr BPC Koeleman, Department of Medical suggest that RANTES is also involved in the process of T- Genetics, University Medical Centre Utrecht, Street 2.112, PO Box cell differentiation. Administration of RANTES and its 85060, Utrecht 3508 AB, The Netherlands. E-mail: [email protected] interaction with the CCR5 receptor bias the T-helper Received 12 May 2006; revised 15 June 2006; accepted 15 June 2006; response toward a T-helper type 1 (Th1)-associated published online 20 July 2006 cytokine profile.18,19 A RANTESÀ/À mouse model showed Association of RANTES with T1D A Zhernakova et al 545 decreased T-cell proliferation and impaired production of 95% CI: 0.45–0.91; P ¼ 0.01) to affected children (Table 1). Th1 cytokines IL-2 and interferon-g.20 The transmission of the rare rs2280788*G allele was also RANTES is located in a cluster with several CC decreased, but failed to reach significance (Table 1). To chemokine genes on 17q12. Three RANTES single- confirm our findings, we tested the same SNPs in a nucleotide polymorphisms (SNPs) in the promoter case–control design using 350 T1D cases and 540 (rs2280788: À28*C/G and rs2107538: À403*G/A) and in controls. We observed a significant decrease in frequency the first intron (rs2280789: Int1.1*T/C) have been shown of rs4251719*A (OR ¼ 0.70; 95% CI: 0.53–0.92; P ¼ 0.009), in different studies to modify the transcriptional activity rs2306630*A (OR ¼ 0.74; 95% CI: 0.56–0.98; P ¼ 0.03) and of RANTES.7,21,22 However, establishing which of these rs2107538*A (OR ¼ 0.79; 95% CI: 0.63–0.99; P ¼ 0.04) in SNPs has functional consequences is complicated by the T1D cases compared to controls (Table 2). high linkage disequilibrium (LD) between them. We then tested whether any particular RANTES haplo- Given the strong involvement of RANTES in the type was associated with T1D. Three frequent haplotypes immune process, we decided to perform a genetic were observed in both cases and controls that together analysis of the RANTES gene region in T1D. To comprise 97% of all haplotypes. As all three haplotypes assess the functional significance of the genetic variants, contained the rs2280788*C allele, the rs2280788 SNP we tested whether the serum level of RANTES was was omitted from the haplotype analysis. Transmission dependent on the underlying genotypes. of the rs4251719*A–rs2306630*A–rs2107538*A haplotype (further referred to as haplotype A–A–A) to T1D offspring was decreased (OR ¼ 0.55; 95% CI: 0.37–0.83; Results P ¼ 0.003). The frequency of this A–A–A haplotype was Haplotype structure of the RANTES region also decreased in T1D cases compared to controls To comprehensively test RANTES for association with (OR ¼ 0.74; 95% CI: 0.55–0.98; P ¼ 0.03; Table 4). The T1D, we first investigated the LD structure of the frequency of the rs4251719*G–rs2306630*G–rs2107538*A RANTES gene (www.hapmap.org).23 We observed haplotype (G–G–A) was similar in cases and controls that RANTES is located in an extended block of strong and transmission of this haplotype was not distorted LD and that two SNPs from the block is sufficient to (Tables 3 and 4). The overall haplotype association tag all the haplotype information (data not shown; was also significant (P ¼ 0.01 and P ¼ 0.02 in TDT and available at http://humgen.med.uu.nl/publications/ the case–control study, respectively; Tables 3 and 4). zhernakova2006_1.html). Next, we assessed whether the haplotype structure in a Association with celiac disease Dutch population is similar to the CEHP families included The positive association found in T1D prompted us to in HAPMAP. In 90 healthy controls that were picked at investigate these RANTES polymorphism in another random from the whole control group, we determined that autoimmune disease, celiac disease (CD). We therefore SNPs rs2306630 and rs3817655 were mutually redundant, genotyped RANTES SNPs rs2306630, rs2280788 and meaning that only one of this pair needed to be used for testing. Thus, only three SNPs – rs4251719, rs2306630 and Table 1 TDT results in T1D families (n ¼ 218) rs2107538 – are informative for the RANTES gene region. Although the infrequent rs2280788 SNP did not differ SNP Allele T NT %T OR (95% CI) P-values between haplotypes, we included this SNP in our study owing to its possible functionality. Consequently, four rs4251719 A 47 79 37.3 0.55 (0.37–0.81) 0.002 SNPs (rs4251719, rs2306630, rs2280788 and rs2107538) G 366 334 52.3 1 (Ref.) were tested in further studies. rs2306630 A 46 73 38.7 0.59 (0.40–0.87) 0.007 G 364 337 51.9 1 (Ref.) rs2280788 G 9 18 33.3 0.51 (0.23–1.14) 0.08 Association studies C 390 381 50.6 1 (Ref.) All four SNPs were tested for association with trans- rs2107538 A 64 92 41.0 0.64 (0.45–0.91) 0.01 mission disequilibrium test (TDT) in our cohort of 218 G 340 312 52.1 1 (Ref.) families. We observed a significant decrease in transmission of the minor alleles of rs4251719*A (OR ¼ 0.55; Abbreviations: CI, 95% confidence interval; NT, non-transmitted; 95% CI: 0.37–0.81; P ¼ 0.002); rs2306630*A (OR ¼ 0.59; OR, odds ratio; T, transmitted; T1D, type 1 diabetes; TDT, 95% CI: 0.40–0.87; P ¼ 0.007) and rs2107538*A (OR ¼ 0.64; transmission disequilibrium test.

Table 2 Allele and genotype frequencies of the SNPs in the RANTES region in T1D (n ¼ 350) and control (n ¼ 540) groups

SNP Allele Genotypes T1D cases Genotypes controls MAF OR (95% CI) P-values

1/2 11 12 22 11 12 22 Case Control rs4251719 A/G 4 (1.2) 76 (22.4) 259 (76.4) 10 (1.9) 159 (30.2) 358 (67.9) 0.12 0.17 0.70 (0.53–0.92) 0.009 rs2306630 A/G 5 (1.5) 75 (22.1) 259 (76.4) 8 (1.5) 159 (29.6) 370 (68.9) 0.13 0.16 0.74 (0.56–0.98) 0.03 rs2280788 C/G 316 (96.0) 13 (4.0) 0 (0.0) 499 (95.0) 25 (4.8) 1 (0.2) 0.02 0.03 0.79 (0.41–1.53) 0.4 rs2107538 A/G 10 (3.0) 100 (30.2) 221 (66.8) 17 (3.2) 204 (38.2) 313 (58.6) 0.18 0.22 0.77 (0.61–0.99) 0.04

Abbreviations: CI, confidence interval; MAF, minor allele frequency; OR, odds ratio; SNP, single-nucleotide polymorphism; T1D, type 1 diabetes.

Genes and Immunity Association of RANTES with T1D A Zhernakova et al 546 Table 3 Transmission of RANTES haplotypes to affected offspring 60000.00 (n ¼ 218)

rs4251719– T NT %T OR (95% CI) P-values Overall 50000.00 rs2306630– P-value rs2107538 40000.00

A–A–A 43 72 37.4 0.55 (0.37–0.83) 0.003 30000.00 G–G–A 16 13 55.2 1.1 (0.53–2.31) 0.8 0.01 G–G–G 325 296 52.3 Ref. Ref. 20000.00

Abbreviations: CI, confidence interval; NT, non-transmitted; OR, 10000.00 odds ratio; T, transmitted. 0 controls patients Table 4 Frequency of RANTES haplotype in T1D cases (n ¼ 350) p=0.01 p=0.09 and controls (n ¼ 540) Figure 1 Correlation of RANTES serum level with the presence or ¼ rs4251719– Cases Controls OR P-values Overall absence of haplotype 2. In the controls (n 14), two individuals rs2306630– (%) (%) (95% CI) P-value carried haplotype 2 (white), 12 were non-carriers (black); in the ¼ rs2107538 patients (n 15), there were five carriers (white) and 10 non-carriers (black). P-value was calculated with Student’s t-test by comparing the RANTES serum level in carriers and non-carriers of the A–A–A 82 (12.5) 169 (16.8) 0.74 (0.55–0.98) 0.03 haplotype 2. G–G–A 36 (5.5) 55 (5.3) 0.99 (0.65–1.53) 0.9 0.02 G–G–G 537 (81.9) 810 (77.8) Ref. Ref. rs2306630*A and rs2107538*A are associated with protec- Abbreviations: CI, confidence interval; OR, odds ratio; T1D, type 1 tion from T1D. We further show that these alleles are diabetes. related to decreased serum protein levels. This implies that RANTES is involved in the pathogenesis of T1D, rs2107538 in a group of 310 CD patients and compared presumably owing to its key role in the process of them with controls. The allele distribution of all polarization, activation and differentiation of T cells. three SNPs in celiac patients was not significantly A number of molecular mechanisms can explain different from that in controls (Supplementary Table 1, the association between the RANTES gene and T1D: also available online at http://humgen.med.uu.nl/ (i) binding of RANTES to its receptor CCR5 leads to the publications/zhernakova2006_1.html). activation of Janus kinases (JAK).24,25 Remarkably, the administration of JAK3 inhibitor JANEX1 delays Relation to RANTES serum level the onset of autoimmune diabetes in non-obese diabetic 26 To investigate the functional relevance of the RANTES (NOD) mice. (ii) RANTES is one of the activation genotype, we tested for correlation between serum level factors of Erk and p38 members of the mitogen-activated and genotypes in an independent cohort of 15 recent protein kinase (MAPK) family that are involved in 17,25,27,28 onset T1D patients and 14 age-matched controls. regulating cell proliferation and differentiation. The patients and controls were genotyped for the SNPs Interestingly, p38 is considered to be a master switch rs2280788, rs2107538 and rs2306630. Only three patients between benign and destructive insulitis and therefore 29 and one control were heterozygous for the rs2280788*G provokes the development of T1D. Inhibition of p38 allele. This SNP was excluded from the analysis because MAPK leads to the inhibition of Th1 immunity and 29 of its low frequency. prevents NOD mice from developing diabetes. It is Carriers of the T1D-associated alleles, rs2107538*A therefore conceivable that a decreased level of RANTES and rs2306630*A (A–A haplotype), had lower levels could offer protection from development of T1D via of RANTES compared to non-carriers (Figure 1). reduced activation of one of these pathways. The mean level of RANTES was significantly lower in Genetic associations of RANTES SNPs with asthma, control carriers for the A–A haplotype (mean 25456.37 atopic dermatitis, sarcoidosis, multiple sclerosis and 2841.1 pg/ml) than in non-carriers (44274.672617.4 pg/ other inflammatory and autoimmune diseases have been 5–12 ml) (P ¼ 0.01). In patient carriers and non-carriers of the reported. Therefore, RANTES could be considered a A–A haplotype, the RANTES serum concentrations gene generally predisposing to autoimmune disease. To were 27 129.873044.1 and 35 591.973369.2 pg/ml, re- test this hypothesis, we investigated RANTES SNPs spectively (P ¼ 0.09). When patients and controls were rs2306630, rs2280788 and rs2107538 in a group of 310 CD combined, the difference in RANTES serum level patients. Although RANTES genotypes did not differ between carriers and non-carriers of the protective significantly between patients and controls (Supplemen- haplotype reached significance (P ¼ 0.003), suggesting tary Table 1), our microarray studies on intestinal that carriers of the protective A–A haplotype have a biopsies indicated increased levels of RANTES in the lower RANTES serum level than non-carriers. intestinal biopsies of CD patients compared to controls (B Diosdado, personal communication). The increase in the level of RANTES correlated with the severity of the Discussion morphological changes in the intestine. Despite a lack of genetic association for RANTES in CD, the fact that the We demonstrate that both in families and in a case– levels of RANTES are increased in patient biopsies, and control cohort, the minor alleles of SNPs rs4251719*A, in combination with the genetic association with

Genes and Immunity Association of RANTES with T1D A Zhernakova et al 547 RANTES in other autoimmune diseases, renders this children was diagnosed according to the International gene a potentially relevant candidate biological marker Society of Pediatric and Adolescent Diabetes (ISPAD) for autoimmune disease. criteria. All these patients were diagnosed at age 17 years Increased protein levels of RANTES in the primary or younger (median 8.7 years, range 1–17 years); 159 sites of inflammation have been reported in different were girls. Both parents were available for 218 of these autoimmune diseases. RANTES levels are increased in patients, so that we could perform a family-based the cerebrospinal fluid of patients suffering active attacks association study on them by means of a TDT. of multiple sclerosis.14 RANTES levels are also increased The control group consisted of 540 independent in allograft rejection,14 in patients with system lupus Dutch individuals, comprising 180 healthy spouses of erythematosis,30 in the synovium of patients with non-diabetic patients included in different (not immune- rheumatoid arthritis31 and in the granulomas in Crohn’s mediated) studies, and 360 healthy blood donor controls, disease.32 Similarly, we here report that the RANTES as described previously.38 Written informed consent was alleles associated with lower serum level of the protein obtained from all participants. are associated with protection from T1D. Serum levels of RANTES were measured in 15 T1D Investigating the functionality of RANTES SNPs is patients and 14 matched controls from the REMTrial complicated by the presence of high LD in this region. project by double-sandwich enzyme-linked immunosor- The transcriptional activity of two promoter SNPs bent assay, as described previously.39 The detection level (rs2280788: À28*C/G and rs2107538: À403*G/A) and an for RANTES was set at 14 pg/ml. SNP in intron 1 (rs2280789: Int1.1*T/C) has been investi- A total of 310 independent CD patients were studied. gated.7,21,22 An et al.22 showed that transcription activity The diagnosis of CD was made according to the revised was mainly regulated by the Int1.1 genotype. Int1.1*C European Society of Paediatric Gastroenterology and allele strongly downregulates the transcriptional activity Nutrition (ESPGAN) criteria.40 In addition, the intestinal of RANTES, whereas the À403*G/A SNP has no effect on biopsies on which the initial diagnoses were based transcription and the À28*G allele shows a modest were re-evaluated for all patients by one experienced upregulation.22 We have investigated the SNP rs2306630, pathologist. Only patients with a Marsh III lesion were which, according to the HAPMAP data, is a perfect proxy included in our study. Of the 310 CD patients, 214 were of the Int1.1 SNP (where the Int1.1*C allele could be tagged female and 96 were male patients. The average age by the rs2306630*G allele and the Int1.1*T allele corre- at diagnosis of CD patients was 34.3 years (range from 1 sponds to the rs2306630*A allele). We observed that the to 83 years). haplotype 2, which is tagged by the rs2306630*A allele, is associated with a lower protein level of RANTES. Our SNP selection results therefore corroborate An et al.’s22 findings. The RANTES gene is located in a region of strong LD of RANTES functions via the CC receptors, mainly CCR5, B155.5 kb extent and it is limited by SNPs rs4796105 and and the association of T1D with the functional variant in rs9899870. We selected two SNPs (rs2306630 and the CCR5 gene (CCR5D32) has been reported pre- rs3817655) that could tag all the frequent haplotypes in viously.33,34 We therefore considered testing for epistasis the block according to the HAPMAP data. We also between RANTES and CCL5 genetic variants. However, included three additional SNPs (rs4251719, rs2280788 the observed frequency of the CCR5D32 variant in our and rs2107538), two of which were located in the patient cohort was too low to provide conclusive promoter region of RANTES (rs2280788 and rs2107538) evidence for interactions between these genes. and have previously been reported to influence the We propose RANTES as a functional candidate gene protein level of RANTES. One additional previously for T1D. However, as RANTES is located in an extended reported functional SNP – Int1.1 (rs2280789) – was typed block of high LD, the observed genetic association in the HAPMAP project and, according to the HAPMAP could be owing to variants in other proximal genes. data, could be replaced by rs2306630 owing to the strong The block of strong LD includes six known and predicted LD and the identical allele frequency. genes (MMP28, TAF15, FLJ32830, CCL5, LOC440427 and RAD52B). Among these, the MMP28 (the epilysin Genotyping member of matrix metalloproteinase (MMP)) could serve SNPs rs2306630, rs4251719, rs3817655 and rs2107538 as an alternative functional candidate for predisposition (À403*G/A) were genotyped using Taqman assays- to autoimmune disease. The MMP28 gene is important on-demand (Applied Biosystems, 2910 AH Nieuwerkerk for tissue remodelling and wound repair. Interestingly, a/d IJssel, The Netherlands) (assay numbers the level of several MMP genes is reported to be C_26924101_10, C_3143173_10, C_2957192_10 and influenced by RANTES stimulation.35–37 The CC chemo- C_15874407_10, respectively). The rs2280788 (À28*C/G) kine genes located telomeric of RANTES are also in SNPs was genotyped using Taqman assay-by-design relatively strong LD with RANTES, and therefore cannot (Applied Biosystems). Assays were performed according be excluded. A detailed study of this region is therefore to the manufacturer’s specifications. Genotypes were necessary to elucidate the contribution of RANTES to the analyzed using a TaqMan 7900HT (Applied Biosystems). observed association. The DNA samples were processed in 384-well plates. Each plate contained eight negative controls and 16 genotyping controls (four duplicates of four different Subjects and methods CEPH samples). In the controls, the frequency of all the SNPs was in Subjects Hardy–Weinberg (HW) proportions, except the A total of 350 patients with juvenile onset T1D were rs2107538 (P ¼ 0.02), which was most likely owing to a included in the case–control study. T1D in all the low number of individuals homozygous for the minor

Genes and Immunity Association of RANTES with T1D A Zhernakova et al 548 allele. To exclude possible genotyping mistakes, we 8 Takada T, Suzuki E, Ishida T, Moriyama H, Ooi H, Hasegawa typed this SNP in the additional cohort of 360 healthy T et al. Polymorphism in RANTES chemokine promoter affects individuals. The allelic and genotypes distribution were extent of sarcoidosis in a Japanese population. Tissue Antigens in HW proportions in the total control group (P ¼ 0.35). 2001; 58: 293–298. 9 Leung TF, Tang NL, Lam CW, Li AM, Fung SL, Chan IH et al. Data analysis RANTES GÀ401A polymorphism is associated with allergen The haplotype structure and LD in the region was sensitization and FEV1 in Chinese children. Respir Med 2005; 99: 216–219. investigated using the HAPMAP data (www.hapmap. 41 42 10 Kozma GT, Falus A, Bojszko A, Krikovszky D, Szabo T, Nagy org) with the Haploview application. Allele and A et al. Lack of association between atopic eczema/dermatitis genotype distribution in cases and controls were com- syndrome and polymorphisms in the promoter region of pared using the COCAPHASED module of the UN- RANTES and regulatory region of MCP-1. Allergy 2002; 57: PHASED statistical package.43 Association in the T1D 160–163. families was tested using the TDT phase module of the 11 Makki RF, al Sharif F, Gonzalez-Gay MA, Garcia-Porrua C, same package.43 Haplotypes association were estimated Ollier WE, Hajeer AH. RANTES gene polymorphism in using the UNPHASED package.43 HW equilibrium was polymyalgia rheumatica, giant cell arteritis and rheumatoid tested by comparing the expected and observed geno- arthritis. Clin Exp Rheumatol 2000; 18: 391–393. types in 2 Â 3 w2 table. ORs were calculated and the 12 Gade-Andavolu R, Comings DE, MacMurray J, Vuthoori RK, CIs were approximated using Woolf’s method with Tourtellotte WW, Nagra RM et al. 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