Polymorphisms in the IL2RA and IL2RB Genes in Inflammatory Bowel Disease Risk

Abstract

Associations with different autoimmune diseases of polymorphisms in genes encoding the IL2RA and IL2RB subunits (located in 10p15 and 22q13, respectively), were identified through genome-wide studies. Polymorphisms in these two genes were studied in 107 inflammatory bowel disease (IBD) patients (39 Crohn's disease [CD] and 68 ulcerative colitis [UC]) and in 162 ethnically healthy controls from Tunisia (Sfax). Two of the 15 IL2RA single-nucleotide polymorphisms (SNPs) genotyped (rs4749924 and rs706778) were significantly associated with UC (p_corr=0.018 and 0.048, respectively), but no evidence of association with CD was observed. The IL2RA GTCT haplotype was also more frequent in UC patients compared to controls (2.6% vs. 0%; p=0.002). One of the 6 IL2RB SNPs genotyped (rs743776) was significantly associated with CD (p_corr= 0.039), but no evidence of association with UC was observed. No significant association between IL2RB haplotypes was observed among investigated groups. Our study identified markers in the IL2RA and IL2RB genes that are significantly associated with UC and CD, respectively. Our results supporting IL2RA and IL2RB as promising candidate genes for IBD and suggesting a potential role of IL2R in the pathogenesis of IBD, likely involves regulatory T cells.

Introduction

Inflammatory bowel disease (IBD) is a chronic condition characterized by recurrent inflammation of intestinal mucosa, and results from aberrant regulation of the mucosal immune system in genetically susceptible individuals. The etiology of IBD involves a complex interaction of genetic, environmental, and immunomodulatory factors. Two major forms of chronic mucosal inflammation have been defined. In Crohn's disease (CD), the whole gastrointestinal tract may be affected, although the most frequent site of inflammation is the terminal ileum, whereas in ulcerative colitis (UC), the mucosal inflammation typically affects the colon (Kaser et al., 2010).

Recently, genome-wide association studies (GWAS) together with meta-analysis of GWAS findings involving IBD (Yamazaki et al., 2005; Duerr et al., 2006; Hampe et al., 2007; The Wellcome Trust Case Control Consortium, 2007; Barrett et al., 2008; Fisher et al., 2008; Franke et al., 2008, 2010a; Asano et al., 2009; Silverberg et al., 2009; McGovern et al., 2010) unearthed several loci conferring risk.

Some of these IBD susceptibility genes are shared with other autoimmune diseases (The Wellcome Trust Case Control Consortium, 2007), probably reminiscent of the higher risk of other immune-mediated conditions among IBD patients compared with controls (Cohen et al., 2008). This genome-wide methodology revealed the importance of the IL2RA and IL2RB genes in several autoimmune diseases such as rheumatoid arthritis (RA) (The Wellcome Trust Case Control Consortium, 2007), multiple sclerosis (Hafler et al., 2007), type 1 diabetes (T1D) (The Wellcome Trust Case Control Consortium, 2007), and celiac disease (van Heel et al., 2007).

The IL2RA and beta (IL2RB) chains, in conjunction with the common gamma chain (IL2RG), form the heterotrimeric high-affinity cell surface receptor for IL-2 (Burchill et al., 2007), a cytokine with pronounced effects on proliferation and expansion of both antigen-specific clones of CD4+ and CD8+ T cells. This cytokine stimulates differentiation to Th1 and Th2 subsets, induces apoptosis of activated T lymphocytes, and plays a nonredundant role in the development of CD4+ CD25+ Tregs (Burchill et al., 2007). In CD8+ cells, IL-2 augments the cytotoxic activity and activates proliferation of memory CD8+ cells (Gaffen and Liu, 2004). The mice lacking either IL-2 or the α- or β-chain of the IL-2R develop a hyperproliferative disorder and show signs of autoimmunity (Sadlack et al., 1993; Suzuki et al., 1995).

Published work empowered to detect general autoimmunity susceptibility loci at the genomic level has provided prime candidate polymorphisms that can be studied in smaller populations and in specific diseases. This will contribute information for meta-analysis studies of specific polymorphisms in autoimmune diseases. Holding a limited sample of IBD patients from Tunisia in Northern Africa, we aimed to study the genetic epidemiology of polymorphisms in specific genes previously associated to other autoimmune diseases, namely, IL2RA and IL2RB genes.

Patients and Methods

Patients

Blood samples were obtained from 107 IBD patients (68 with UC, 39 with CD) diagnosed at the Department of Gastroenterlogy of Hedi Chaker University Hospital (Sfax, Tunisia) from March 2004 to May 2008. All patients were from Tunisian descent (North Africa). The diagnosis was based on standard clinical, radiographic, endoscopic, and histopathological criteria (Podolsky, 1991). Table 1 summarizes clinical data. Patients with CD were assessed according to the Montreal classification (Silverberg et al., 2005) based on the age at diagnosis, localization, and behavior of disease. In patients with UC, anatomic localization was also determined according to the Montreal classification, using the criteria ulcerative proctitis, left sided, and extensive. As unaffected controls (HC), 162 healthy Tunisian subjects with no history of digestive system disease, unrelated to each other or to the patients were recruited in this study.

Table 1.

Clinical Description of Crohn's Disease and Ulcerative Colitis Patients

	Crohn's disease	Ulcerative colitis	Total (IBD)
Patients (n)	39	68	107
Sex (male/female)	24/15	33/35	57/50
Age (years), mean±SD	39.4±13.8	40.5±12.4	40.1±13.2
Pathology location
Ileum	14
Colon	11
Ileocolon	14
Pancolitis		35
Left-sided		26
Proctitis		7
Extraintestinal manifestations	18	13	31

IBD, inflammatory bowel disease.

The study was approved by the local ethics committee and all enrolled patients provided their informed consent to participate.

Methods

Genotyping methods

Genomic DNA was extracted from whole blood samples using a standard proteinase K digestion and phenol/chloroform extraction procedure. Genotyping was performed using primer extension chemistry and mass spectrometric analysis (iPlex assay; Sequenom, San Diego, CA) on the Sequenom MassArray at the Instituto Gulbenkian de Ciência, Oeiras, Portugal. We genotyped 15 single-nucleotide polymorphisms (SNPs) in IL2RA and 6 SNPs in IL2RB. The selected reference SNPs were retrieved from the HapMap database and the mapping information obtained from the db SNP built 126 (Table 2).

Table 2.

Gene Positioning and Alleles of Selected Single-Nucleotide Polymorphisms

Gene name	SNP	Position	Allele	HWE
IL2RA	rs10795791	6108340	A/G	5.01E-01
	rs10905669	6092093	C/T	6.23E-01
	rs11256369	6066200	C/G	7.67E-01
	rs11597367	6107534	A/G	2.56E-01
	rs2076846	6063253	A/G	8.39E-01
	rs2104286	6099045	T/C	7.76E-01
	rs4147359	6108439	G/A	6.86E-01
	rs4749924	6082396	A/C	5.04E-01
	rs706778	6098949	C/T	7.43E-01
	rs706781	6086385	C/T	6.54E-01
	rs7072398	6079846	G/A	4.21E-01
	rs7072793	6106266	T/C	6.12E-01
	rs7090530	6110875	C/A	7.51E-01
	rs791587	6088699	A/G	2.98E-01
	rs9663421	6055604	C/T	5.74E-01
IL2RB	rs2281094	37535471	C/T	1.36E-01
	rs228937	37520971	G/T	8.22E-01
	rs3218292	37533530	G/C	7.48E-01
	rs3218312	37530332	A/G	8.42E-01
	rs743776	37551487	T/C	1.22E-01
	rs84460	37525731	A/G	1.78E-01

HWE, Hardy-Weinberg equilibrium; SNP, single-nucleotide polymorphisms.

The assay design was performed according to the manufacturer's instructions, whereby the genomic sequence containing the SNP is amplified by multiplex PCR. The amplified product was cleaned using shrimp alkaline phosphatase and used for allele-specific primer extension reaction according to the MassEXTEND protocol. The reaction mixture was then spotted onto a SpectroCHIP microarray and subjected to MALDI-TOF mass spectrometry. SpectroTYPER software identifies the SNP-specific peaks and automatically assigns the genotype calls.

Data analysis

Quality control criteria excluded SNPs with a call rate lower than 95% in cases and controls. Genotype frequencies were in Hardy-Weinberg equilibrium for all the typed SNPs (p>0.05) of the Tunisian control population. Case-control association analysis was performed for each SNP. Calculations of allelic and genotypic associations of SNPs with susceptibility to IBD were performed using a home-made program written in R language (www.r-project.org). This is a simple program that computes classical allelic and genotypic chi-square tests in an automated manner to avoid computing tests for each SNP one by one. Bonferroni's correction was carried out when SNP analysis yielded significant results. Relative risks were calculated as odds ratio (OR) using 2×2 contingency tables.

Haplotype association analysis was performed using the Haploview program (v. 4.0) (www.broad.mit.edu/mpg/haploview/). Haplotype blocks were defined by solid spine rule and pairwise SNP linkage disequilibrium (LD) coefficients were estimated. For all the analyses, differences were considered statistically significant when the p value was less than or equal to 0.05.

Results

IL2RA variants and IBD

First, we compared the IL2RA genotype and allele frequencies between the UC, CD, and control subjects. Among the 15 SNPs genotyped in this study, 2 SNPs (rs4749924 and rs706778) had statistically significant UC association. In contrast, no evidence of association with CD was found for any SNP in the IL2RA loci.

As shown in Table 3, the CC genotype at rs4749924 SNP was significantly associated with UC (OR=3.33, 95% confidence interval [CI]=1.36-8.15, p=0.006, p_corr=0.018). The CT genotype of the IL2RA variant rs706778 showed a protective effect when UC patients were compared with healthy controls (OR=0.48, 95% CI=0.26-0.88, p=0.016, p_corr=0.048).

Table 3.

Genotypic and Allelic Association Tests of IL2RA and IL2RB SNP Polymorphisms with CD and UC

		Controls	CD			UC
GENE/SNP	Genotypes/alleles	N (%)	N (%)	p-Value^a (pcorr)^b	OR (95% CI)	N (%)	p-Value^a (Pcorr)^b	OR (95% CI)
IL2RA
rs10795791	AA	59 (37)	12 (31)	0.42	0.74 (0.35-1.57)	27 (41)	0.58	1.18 (0.65-2.13)
	AG	78 (50)	20 (51)	0.85	0.94 (0.47-1.89)	22 (34)	0.031 (0.093)	0.52 (0.28-0.95)
	GG	20 (13)	7 (18)	0.39	1.50 (0.58-3.85)	16 (25)	0.029 (0.087)	2.24 (1.07-4.66)
	A	196 (63)	44 (56)	0.32	0.78 (0.47-1.29)	76 (58)	0.47	0.85 (0.56-1.29)
rs10905669	CC	98 (63)	27 (71)	0.32	1.48 (0.68-3.20)	43 (66)	0.6	1.18 (0.64-2.16)
	TC	54 (34)	8 (21)	0.11	0.51 (0.22-1.19)	19 (29)	0.46	0.79 (0.42-1.48)
	TT	5 (3)	3 (8)	0.18	2.61 (0.59-11.42)	3 (5)	0.6	1.47 (0.68-6.34)
	C	250 (80)	62 (82)	0.7	1.13 (0.60-2.15)	105 (81)	0.82	1.08 (0.64-1.80)
rs11256369	GG	25 (17)	8 (21)	0.52	1.33 (0.55-3.25)	17 (27)	0.084	1.85 (0.92-3.73)
	GA	64 (43)	12 (32)	0.21	0.62 (0.29-1.32)	24 (38)	0.53	0.83 (0.45-1.51)
	AA	61 (40)	18 (47)	0.45	1.31 (0.64-2.69)	22 (35)	0.43	0.78 (0.42-1.44)
	A	186 (62)	48 (63)	0.85	1.05 (0.62-1.77)	68 (54)	0.12	0.72 (0.47-1.09)
rs11597367	AA	77 (50)	22 (56)	0.47	1.29 (0.64-2.63)	32 (48)	0.83	0.94 (0.53-1.68)
	AG	59 (38)	14 (36)	0.78	0.90 (0.43-1.87)	23 (35)	0.62	0.86 (0.47-1.57)
	GG	18 (12)	3 (8)	0.47	0.63 (0.18-2.26)	11 (17)	0.31	1.51 (0.67-3.41)
	A	213 (69)	58 (74)	0.37	1.29 (0.74-2.27)	87 (66)	0.5	0.86 (0.56-1.33)
rs2076846	AA	86 (55)	27 (69)	0.14	1.86 (0.88-3.93)	38 (58)	0.7	1.12 (0.63-2)
	AG	60 (38)	10 (26)	0.12	0.56 (0.25-1.23)	21 (32)	0.37	0.75 (0.41-1.39)
	GG	11 (7)	2 (5)	0.91	0.72 (0.15-3.38)	7 (10)	0.37	1.57 (0.58-4.26)
	A	232 (74)	64 (82)	0.25	1.62 (0.86-3.04)	97 (73)	0.93	0.98 (0.62-1.55)
rs2104286	TT	5 (3)	2 (5)	0.51	1.73 (0.32-9.26)	4 (6)	0.34	1.92 (0.50-7.38)
	TC	43 (28)	10 (27)	0.94	0.97 (0.43-2.18)	17 (25)	0.73	0.89 (0.47-1.72)
	CC	108 (68)	25 (68)	0.84	0.93 (0.43-2)	46 (69)	0.93	0.97 (0.52-1.81)
	C	259 (83)	60 (81)	0.69	0.88 (0.46-1.68)	109 (81)	0.67	0.89 (0.53-1.51)
rs4147359	GG	13 (8)	6 (17)	0.13	2.17 (0.76-6.17)	6 (10)	0.79	1.14 (0.41-3.15)
	GA	59 (38)	13 (36)	0.81	0.91 (0.43-1.93)	22 (35)	0.64	0.86 (0.47-1.59)
	AA	82 (53)	17 (47)	0.52	0.79 (0.38-1.63)	35 (55)	0.76	1.1 (0.61-1.98)
	G	223 (72)	47 (65)	0.23	0.72 (0.42-1.24)	92 (93)	0.89	1.03 (0.65-1.64)
rs4749924	AA	94 (60)	21 (55)	0.61	0.83 (0.41-1.69)	34 (52)	0.3	0.74 (0.41-1.32)
	AC	53 (34)	14 (37)	0.72	1.14 (0.55-2.39)	19 (29)	0.51	0.81 (0.43-1.52)
	CC	10 (6)	3 (8)	0.74	1.26 (0.33-4.82)	12 (18)	0.006(0.018)	3.33 (51.36-8.15)
	C	73 (23)	20 (26)		1.18 (0.66-2.09)	43 (33)	0.03 (0.06)	1.63 (1.04-2.56)
rs706778	CC	51 (33)	12 (32)	0.84	0.92 (0.43-1.98)	30 (46)	0.088	1.67 (0.92-3.01)
	CT	73 (48)	18 (47)	0.97	0.99 (0.48-2.01)	20 (30)	0.016 (0.048)	0.48 (0.26-0.88)
	TT	29 (19)	8 (21)	0.77	1.14 (0.47-2.74)	16 (24)	0.37	1.37 (0.68-2.74)
	C	175 (57)	42 (55)	0.76	0.92 (0.56-1.53)	80 (61)	0.51	1.15 (0.76-1.75)
rs706781	CC	7 (5)	2 (5)	0.90	1.13 (0.22-5.65)	7 (11)	0.09	2.47 (0.83-7.36)
	CT	58 (38)	9 (23)	0.068	0.49 (0.22-1.11)	16 (24)	0.05	0.52 (0.27-1)
	TT	88 (57)	28 (72)	0.084	1.88 (0.87;4.05)	43 (65)	0.29	1.38 (0.76-2.51)
	T	234 (76)	65 (83)	0.16	1.54 (0.8-2.95)	102 (77)	0.86	1.05 (0.64-1.70)
rs7072398	GG	47 (30)	14 (36)	0.5	1.29 (0.61-2.69)	29 (44)	0.05	1.75 (0.97-3.17)
	AG	72 (47)	16 (41)	0.54	0.80 (0.39-163)	27 (41)	0.44	0.78 (0.44-1.39)
	AA	36 (23)	9 (23)	0.98	0.99 (0.43-2.28)	11 (15)	0.17	0.65 (0.31-1.37)
	A	144 (46)	34 (44)	0.65	1.12 (0.68-1.85)	47 (36)	0.035 (0.07)	1.57 (51.03-2.39)
rs7072793	TT	22 (15)	6 (17)	0.75	1.17 (0.44-3.14)	15 (23)	0.11	1.79 (0.86-3.74)
	TC	76 (50)	19 (53)	0.79	1.1 (0.53-2.28)	23 (36)	0.05	0.55 (0.30-1.01)
	CC	53 (35)	11 (31)	0.61	0.81 (0.37-1.78)	26 (41)	0.44	1.27 (0.69-2.31)
	C	182 (60)	41 (57)	0.61	0.87 (0.52-1.47)	75 (59)	0.74	0.93 (0.61-1.42)
rs7090530	CC	41 (26)	14 (36)	0.2	1.61 (0.77-3.39)	23 (35)	0.17	1.54 (0.83-2.86)
	CA	77 (48)	16 (41)	0.4	0.74 (0.36-1.51)	24 (36)	0.09	0.61 (0.34-1.1)
	AA	41 (26)	9 (23)	0.72	0.86 (0.38-197)	19 (29)	0.64	1.16 (0.61-2.21)
	C	159 (50)	44 (56)	0.3	1.29 (0.79-2.13)	70 (53)	0.55	1.13 (0.75-1.70)
rs791587	AA	55 (35)	12 (31)	0.59	0.82 (0.38-1.74)	28 (43)	0.28	1.39 (0.77-2.51)
	AG	76 (48)	18 (46)	0.77	0.90 (0.45-1.82)	23 (35)	0.07	0.58 (0.32-1.05)
	GG	25 (7)	9 (23)	0.29	1.57 (0.67-3.71)	14 (22)	0.32	1.44 (0.69-2.98)
	A	186 (60)	42 (54)	0.35	0.79 (0.48-1.30)	79 (61)	0.82	1.05 (0.69-1.59)
rs9663421	CC	76 (49)	18 (49)	0.96	0.98 (0.48-2.02)	30 (45)	0.62	0.87 (0.49-1.54)
	CT	63 (41)	14 (38)	0.75	0.89 (0.43-1.86)	27 (41)	0.97	1.01 (0.56-1.82)
	TT	16 (10)	5 (13)	0.57	1.36 (0.46-3.98)	9 (14)	0.47	1.37 (0.57-3.28)
	C	215 (69)	50 (68)	0.76	0.92 (0.53-1.58)	87 (66)	0.47	0.85(0.55-1.32)
IL2RB
rs2281094	CC	98 (62)	23 (59)	0.76	0.89 (0.44-1.83)	42 (64)	0.77	1.09 (0.6-1.97)
	CT	58 (36)	13 (33)	0.71	0.87 (0.42-1.82)	20 (30)	0.37	0.76 (0.41-1.40)
	TT	3 (2)	3 (8)	0.05	4.33 (0.84-22.36)	4 (6)	0.1	3.35 (0.73-15.43)
	C	254 (80)	59 (76)	0.41	0.78 (0.44-1.40)	104 (79)	0.79	0.94 (0.57-1.54)
rs228937	GG	9 (6)	1 (3)	0.41	0.43 (0.05-3.47)	4 (6)	0.94	1.05 (0.31-3.53)
	GT	54 (35)	12 (31)	0.63	0.83 (0.39-1.77)	32 (48)	0.057	1.76 (0.98-3.16)
	TT	92 (59)	26 (67)	0.41	1.37 (0.65-2.87)	30 (45)	0.057	0.57 (0.32-1.02)
	T	238 (77)	64 (82)	0.32	1.38 (0.73-2.61)	92 (70)	0.11	0.70 (0.44-1.10)
rs3218292	GG	113 (73)	26 (75)	0.8	1.07 (0.47-2.48)	54 (80)	0.24	1.54 (0.77-3.11)
	GC	38 (25)	7 (19)	0.51	0.77 (0.31-1.90)	11 (17)	0.19	0.60 (0.29-1.27)
	CC	4 (3)	2 (6)	0.35	2.29 (0.40-13.02)	2 (3)	0.85	1.16 (0.21-6.50)
	G	264 (85)	59 (84)	0.92	0.93 (0.46-1.91)	119 (89)	0.33	1.38 (0.74-2.57)
rs3218312	AA	11 (7)	2 (5)	0.65	0.70 (0.15-3.31)	8 (12)	0.25	1.74 (0.67-4.56)
	AG	62 (41)	15 (39)	0.83	0.93 (0.45-1.92)	21 (32)	0.18	0.66 (0.36-1.22)
	GG	77 (51)	21 (55)	0.66	1.17 (0.57-2.39)	37 (56)	0.52	1.21 (0.68-2.16)
	G	216 (72)	57 (75)	0.6	1.17 (0.66-2.08)	95 (72)	0.99	1 (0.63-1.58)
rs743776	TT	5 (3)	5 (13)	0.013 (0.039)	4.55 (1.24. 16.61)	6 (9)	0.06	3 (0.88-10.20)
	TC	64 (41)	9 (24)	0.045 (0.14)	0.44 (0.2. 1)	17 (26)	0.028 (0.084)	0.49 (0.26. 0.93)
	CC	86 (56)	24 (63)	0.39	1.38 (0.66-2.86)	43 (65)	0.18	1.50 (0.83-2.73)
	C	236 (76)	57 (75)	0.83	0.94 (0.53-1.68)	103 (78)	0.66	1.11 (0.68-1.81)
rs84460	AA	10 (7)	4 (10)	0.45	1.62 (0.48-5.48)	6 (9)	0.53	1.40 (0.49-4.01)
	AG	72 (47)	11 (28)	0.048 (0.14)	0.44 (0.20-0.94)	25 (36)	0.11	0.65 (0.36-1.16)
	GG	70 (46)	24 (62)	0.11	1.87 (0.91-3.85)	37 (55)	0.21	1.40 (0.79-2.48)
	G	212 (70)	59 (76)	0.35	1.35 (0.76-2.39)	98 (73)	0.47	1.18 (0.75-1.86)

Significant associations are highlighted in bold.

Two-tailed p-values were calculated by homemade program written in R language.

Corrected p-values p_corr—after Bonferroni correction—are shown in brackets.

CI, confidence interval; OR, odds ratio; CD, Crohn's disease; UC, ulcerative colitis.

The significant effects of rs7072398 (A allele, p=0.035), rs10795791 (GG genotype, p=0.029), and rs7073236 (CT genotype, p=0.036) on UC susceptibility, was lost after Bonferroni's correction (Table 3).

IL2RB variants and IBD

No evidence of association with UC was found for any SNP studied in the IL2RB loci.

As shown in Table 3, the TT genotype at rs743776 SNP was significantly associated with CD (OR=4.55, 95% CI=1.24-16.61, p=0.013, p_corr=0.039).

IL2RA and IL2RB haplotype analysis

Then, we measured the LD patterns of the SNPs and identified four LD blocks from the SNPs of IL2RA and one LD block in the SNPs of IL2RB (Fig. 1). To determine whether any specific haplotype would confer a higher risk or protection for UC and CD, we performed the haplotype test of association.

FIG. 1.

Linkage disequilibrium (LD) patterns of the region around the IL2RA and IL2RB genes in the Tunisian population. Spectrum from white to grey displays the D' measure, with darker boxes indicating stronger disequilibrium. Four LD blocks were found in IL2RA, and one LD block was in IL2RB. (A) LD patterns of IL2RA. (B) LD patterns of IL2RB.

The rare haplotype (GTCT) of IL2RA, formed with four SNPs (rs791587, rs10905669, rs706778, and 2104286), was observed to be a risk haplotype, with a frequency of 2.6% in UC patients and 0% in controls (p=0.0020) (Table 4). However, the results of haplotype analysis of IL2RA showed that there was no significant association between these haplotypes for CD patients as compared with control subjects (data not shown).

Table 4.

Haplotype Frequencies Formed by the Four SNPs (rs791587, rs10905669, rs706778, and 2104286) in UC and CD Cases and Healthy Controls

	Frequencies
Haplotype	CD	UC	Controls
ACTT	0.191	0.212	0.207
ACCT	0.348	0.391	0.378
GCCC	0.184	0.174	0.161
GTTT	0.184	0.167	0.195
GTCT	0	0.026^a	0
GCTT	0.072	0.015	0.025
GCCT	0.020	0.015	0.013

p=0.002.

The study of inferred haplotypes in IL2RB did not add any further information neither in UC nor in CD (data not shown).

Discussion

The purpose of this study was to evaluate whether the IL2RA and IL2RB gene polymorphisms may be associated with the risk of IBD in the Tunisian population.

Recently, a strong genetic evidence suggesting for a critical role of IL-2/IL-2RA-dependent regulatory pathway in human autoimmunity was obtained. Findings in humans are supported by studies on an animal model. In fact, mice deficient for IL-2 or IL-2-RA exhibit acute systemic inflammatory disease with clinical symptoms similar to those of UC in humans. The inflammation is accompanied with massive expansion of T and B lymphocytes in nonlymphoid tissues and high serum levels of self-antibodies followed by the death of the animals (Kundig et al., 1993; Sadlack et al., 1993; Willerford et al., 1995). Concordantly, our data reveal evidence of the IL2RA gene in UC risk.

In the context of the studied IL2RA polymorphisms, the strongest genetic effect in our study was attributed to the rs4749924. To our knowledge, the association with this SNP has not been previously described in IBD. However, Fourati et al. (2012) reported a significantly increased incidence of this polymorphism in patients with systemic lupus erythematosus (SLE). Polymorphisms within the gene encoding IL-2RA (rs2104286) were associated with RA and T1D, but not with UC in the Spanish study (Márquez et al., 2009), concordantly with our results.

Furthermore, among the 15 IL2RA SNPs studied, the CT genotype of the rs706778 was found to be protective with regard to our UC cohort as compared with control subjects (OR=0.48; CI=0.26-0.88). However, another study reported a significantly increased incidence of this SNP in patients with DT1 (Kawasaki et al., 2009). The opposing effect associated with this SNP indicates the presence of allelic heterogeneity between T1D and UC. A similar finding was reported for rs11594656 with discrepant effects of the minor allele in SLE and DT1 (Nakanishi and Inoko, 2006; Kawasaki et al., 2008). This phenomenon might reflect genuine immunopathologic differences that distinguish various autoimmune conditions (Dendrou et al., 2009). On the other hand, disparate genetic associations always raise suspicions that a true causative variant is located elsewhere within the LD block. Considering the IL2RA locus, this could be in the neighboring RBM17 gene, for instance.

Other investigated IL2RA SNPs, rs2104286, rs7090530, and rs4147359, did not display any significant association with UC in the current study, but had a significant association with other autoimmune diseases (Lowe et al., 2007; Alcina et al., 2009; Cavanillas et al., 2010; Stahl et al., 2010). Building upon these observations, we confirmed the extended degree of allelic heterogeneity at this autoimmune risk locus.

Our analysis did not detect association with CD susceptibility. Similar to our findings, the genome-wide study carried out by the WTCCC and a very recent study from Spain found no association of IL2RA SNPs with CD (Márquez et al., 2009). In contrast, a genome-wide meta-analysis study confirmed the association between IL2RA polymorphism and CD susceptibility (Franke et al., 2010b). This discrepancy might result from population differences, but we cannot exclude that our negative result is due to the limited sample size.

From the LD analysis, we found that one haplotype (GTCT) of IL2RA was significantly associated with UC in the Tunisian population. These data are novel and have not been reported before. Recently, it has been demonstrated that differences in the surface expression of the IL2RA or CD25 protein are restricted to particular immune cell types and correlated with several haplotypes in the IL2RA region, underscoring the gene-phenotype association between protein levels on specific subsets of primary human immune cell types and autoimmune disease polymorphisms (Dendrou et al., 2009).

As previously reported, mice lacking expression of IL-2RB exhibit severe autoimmunity and they manifest dysregulated T-cell activation and B-cell differentiation (Suzuki et al., 1995, 1997), arguing in favor of a role of the IL2RB gene in the disease. Concordantly, we have shown that the IL2RB gene polymorphism was associated with the risk of CD. In contrast, a Spanish study found no association of IL2RB with CD. The reason for this discrepancy may be an interspecies difference.

In conclusion, this study shows plausible association of the polymorphic IL2RA variants with UC and IL2RB with CD in the Tunisian population. These facts are consistent with a major role for the IL-2/IL-2 receptor pathway in the control of autoimmunity. However, more studies within other populations are necessary to prove the general relevance of these polymorphisms for IBD susceptibility.

Footnotes

Acknowledgments

This work was supported by Ministère de la recherche Scientifique et de la Technologie (MRST) (Tunisia). Genotyping was supported by the Instituto Gulbenkian de Ciência, Oeiras, Portugal. We are grateful to João Costa for providing technical support in genotyping.

Author Disclosure Statement

No competing financial interests exist.

References

Alcina

, Fedetz

, Ndagire

et al. 2009. IL2RA/CD25 gene polymorphisms: uneven association with multiple sclerosis (MS) and type 1 diabetes (T1D) PLoS One, 4:e4137.

Asano

, Matsuhita

, Umeno

et al. 2009. A genome-wide association study identifies three new susceptibility loci for ulcerative colitis in the Japanese population. Nat Genet, 41:1325-1329.

Barrett

, Hansoul

, Nicolae

et al. 2008. Genomewide association defines more than 30 distinct susceptibility loci for Crohn's disease. Nat Genet, 40:955-962.

Burchill

, Yang

, Vang

, Farrar

. 2007. Interleukin-2 receptor signaling in regulatory T cell development and homeostasis. Immunol Lett, 114:1-8.

Cavanillas

, Alcina

, Núñez

et al. 2010. Polymorphisms in the IL2, IL2RA and IL2RB genes in multiple sclerosis risk. Eur J Hum Genet, 18:794-799.

Cohen

, Robinson

Jr , Paramore

et al. 2008. Autoimmune disease concomitance among inflammatory bowel disease patients in the United States, 2001-2002. Inflamm Bowel Dis, 14:738-743.

Dendrou

, Plagnol

, Fung

et al. 2009. Cell-specific protein phenotypes for the autoimmune locus IL2RA using a genotype-selectable human bioresource. Net Genet, 41:1011-1015.

Duerr

, Taylor

, Brant

et al. 2006. A genome-wide association study identifies IL23R as an inflammatory bowel disease gene. Science, 314:1461-1463.

Fisher

, Tremelling

, Anderson

et al. 2008. Genetic determinants of ulcerative colitis include the ECM1 locus and five loci implicated in Crohn's disease. Nat Genet, 40:710-712.

10.

Franke

, Balschun

, Karlsen

et al. 2008. Sequence variants in IL10, ARPC2 and multiple other loci contribute to ulcerative colitis susceptibility. Nat Genet, 40:1319-1323.

11.

Franke

, Balschun

, Sina

et al. 2010a. Genome wide association study for ulcerative colitis identifies risk loci at 7q22and 22q13 (IL17REL) Nat Genet, 42:292-294.

12.

Franke

, McGovern

, Barrett

et al. 2010b. Genome-wide meta-analysis increases to 71 the number of confirmed Crohn's disease susceptibility loci. Nat Genet, 42:1118-1125.

13.

Fourati

, Bouzid

, Abida

et al. 2012. Genetic factors contributing to systemic lupus erythematosus in tunisian patients. J Clin Cell Immunol[Epub ahead of print]10.4172/2155-9899.1000129.

14.

Gaffen

, Liu

. 2004. Overview of interleukin-2 function, production and clinical applications. Cytokine, 28:109-123.

15.

Hafler

, Compston

, Sawcer

et al. 2007. Risk alleles for multiple sclerosis identified by a genome wide study. N Engl J Med, 357:851-862.

16.

Hampe

, Franke

, Rosenstiel

et al. 2007. A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn disease in ATG16L1. Nat Genet, 39:207-211.

17.

Kaser

, Zeissig

, Blumberg

. 2010. Inflammatory bowel disease. Annu Rev Immunol, 28:573-621.

18.

Kawasaki

, Awata

, Ikegami

et al. 2009. Genetic association between the interleukin-2 receptor-alpha gene and mode of onset of type 1 diabetes in the Japanese population. J Clin Endocrinol Metab, 94:947-952.

19.

Kawasaki

, Uga

, Nakamura

et al. 2008. Association between anti-ZnT8 autoantibody specificities and SLC30A8 Arg325Trp variant in Japanese patients with type 1 diabetes. Diabetologia, 51:2299-2302

20.

Kundig

, Schorle

, Bachmann

et al. 1993. Immune responses in interleukin-2-deficient mice. Science, 262:1059-1061.

21.

Lowe

, Cooper

, Brusko

et al. 2007. Large-scale genetic fine mapping and genotype-phenotype associations implicate polymorphism in the IL2RA region in type 1 diabetes. Nat Genet, 39:1074-1082

22.

Márquez

, Orozco

, Martínez

et al. 2009. Novel association of the interleukin 2-interleukin 21 region with inflammatory bowel disease. Am J Gastroenterol, 104:1968-1975.

23.

McGovern

, Gardet

, Törkvist

et al. 2010. Genome wide association identifies multiple ulcerative colitis susceptibility loci. Nat Genet, 42:332-337.

24.

Nakanishi

, Inoko

. 2006. Combination of HLA-A24,DQA1*03, and-DR9 contributes to acute-onset and early complete beta-cell destruction in type 1 diabetes: longitudinal study of residual cell function. Diabetes, 55:1862-1868

25.

Podolsky

. 1991. Inflammatory bowel disease (1) N Engl J Med, 325:928-937.

26.

Sadlack

, Merz

, Schorle

et al. 1993. Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene. Cell, 75:253-261

27.

Silverberg

, Satsangi

, Ahmad

et al. 2005. Toward and integrated clinical, molecular and serological classification of inflammatory bowel disease: report of a working party of the 2005 Monteral World Congress of Gastroenterology. Can J Gastroenterol, 19,Suppl A:5-36

28.

Silverberg

, Cho

, Rioux

et al. 2009. Ulcerative colitis-risk loci on chromosomes 1p36 and 12q15 found by genomewide association study. Nat Genet, 41:216-220.

29.

Stahl

, Raychaudhuri

, Remmers

et al. 2010. Genome-wide association study meta-analysis identifies seven new rheumatoid arthritis risk loci. Nat Genet, 42:508-514

30.

Suzuki

, Duncan

, Takimoto

, Mak

. 1997. Abnormal development of intestinal intraepithelial lymphocytes and peripheral natural killer cells in mice lacking the IL-2 receptor beta chain. J Exp Med, 185:499-505.

31.

Suzuki

, Kundig

, Furlonger

et al. 1995. Deregulated T cell activation and autoimmunity in mice lacking interleukin-2 receptor beta. Science, 268:1472-1476.

32.

The Wellcome Trust Case Control Consortium. 2007. Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature, 447:661-678.

33.

van Heel

, Farnke

, Hunt

et al. 2007. A genome-wide association study for celiac disease identifies risk variants in the region harboring IL2 and IL21. Nat Genet, 39:827-829.

34.

Willerford

, Chen

, Ferry

et al. 1995. Interleukin-2 receptor a chain regulates the size and content of the peripheral lymphoid compartment. Immunity, 3:521-530.

35.

Yamazaki

, McGovern

, Ragoussis

et al. 2005. Single nucleotide polymorphisms in TNFSF15 confer susceptibility to Crohn's disease. Hum Mol Genet, 15:3499-3506.