Abstract
Signal transducer and activator of transcription 6 (STAT6) is a key transcription factor involved in interleukin 4 (IL-4) and IL-13-mediated Th2 response. The STAT6 gene is located on chromosome 12q13.3–14·1 (IBD2 region) and is therefore a positional and functional candidate gene for study in inflammatory bowel disease. We investigated the G2964A polymorphism in the 3′ untranslated region of the STAT6 gene in Dutch patients with inflammatory bowel disease and healthy controls. The G2964A polymorphism in the STAT6 gene was genotyped in 141 unrelated Dutch Caucasian patients with ulcerative colitis, 183 patients with Crohn's disease and 173 healthy individuals by PCR and the amplification-created restriction site method. Patients with Crohn's disease were classified according to the Vienna classification and the patients with ulcerative colitis were classified with the age at onset, extent of disease and colectomy. We did not find significant differences in genotype and allele frequencies of the G2964A polymorphism in the STAT6 gene between ulcerative colitis, Crohn's disease and healthy controls. Subgroups of the patients with Crohn's disease classified according to the Vienna classification and those with ulcerative colitis classified according to age of onset, disease extension and colectomy did not differ in the distribution of this polymorphism. The STAT6 G2964A gene polymorphism is not involved in the overall susceptibility or in determining the phenotype of IBD.
Keywords: Crohn's disease, inflammatory bowel disease, STAT6 gene polymorphism, ulcerative colitis
Introduction
Inflammatory bowel diseases (IBD) are clinically classified as ulcerative colitis (UC), Crohn's disease (CD) and indeterminate colitis. The aetiology of IBD is still unclear and remains under intensive study. IBD manifests as chronic intestinal inflammation due to an exaggerated immune response. An imbalance in the activation of Th1 and Th2 lymphocytes has been found in the intestinal mucosa in IBD [1]. Th1 cytokines including IL-1, IL-2, TNF-α, IL-6, IL-8 and IL-12 promote inflammation while Th2 cytokines down-regulate Th1 responses by secretion of IL-4, IL-10 and IL-13 [2]. IL-4 has multiple functions involved in regulation of CD23 and MHC class II gene expression, B and T cell growth and B cell differentiation into IgE producers and Th0 cell differentiation into Th2 cells [3,4]. The IL-4 receptor (IL-4R) and IL-13R share the IL-4 receptor α-chain encoded by the IL4RA gene and result in the activation of signal transducer and activator of transcription 6 (STAT6) through phosphorylation by Janus kinases 1 and 3 [5,6]. STAT6 forms homodimers, translocates to the nucleus and binds to promoter regions for regulation of transcription. STAT6-binding sites are present in the promoter regions of IL-4 inducible genes [7,8]. STAT6-deficient mice lack a Th2 cytokine response and IgE production, demonstrating that STAT6 is a key transcription factor involved in IL-4 and IL-13-mediated Th2 response [4,9].
The STAT6 gene is located on chromosome 12q13.3–14·1 just within the IBD2 locus, as defined by recent genome-wide linkage studies [10–13]. These studies identified this region to harbour a susceptibility locus for UC and CD. Later studies found some evidence that this region contributes to the susceptibility to UC, but not to CD [14–16]. However, these results were not confirmed in all linkage studies [17–20]. It is interesting that in this region a TaqI polymorphism at codon 352 in exon 8 of the vitamin d-receptor gene was found to be associated with CD [21] while negative results were found for the beta-7 integrin and interferon-γ genes also in this region [22,23]. Two groups of investigators found associations of G2964A genotype in the 3′ untranslated region of the STAT6 gene with asthma and mild-type atopy [24] and nut allergy [25]. Another group reported that a dinucleotide repeat polymorphism in exon 1 of the STAT6 gene was associated with allergic disease [26]. Therefore, STAT6 is a positional and functional candidate gene for studies of susceptibility to IBD.
In the present study, we investigated the STAT6 gene G2964A polymorphism in Dutch Caucasian patients with IBD and healthy controls, and we studied the association between this single nucleotide polymorphism (SNP) and clinical subgroups of the patients.
Subjects and methods
Subjects
One hundred and forty-one patients with UC (male 68, female 73), 183 patients with CD (male 58, female 125) attending the Department of Gastroenterology at the Vrije Universiteit medical centre (VUmc) and 173 healthy controls (male 79, female 94) were studied. The control population consisted of healthy staff and students of the VUmc. All subjects were unrelated Dutch Caucasians. The mean age at onset was 32 ± 14 years for UC and 28 ± 12 years for CD. The age of the control population was 36 ± 12 years. The mean course of the disease was 5·2 ± 6·6 years for UC and 5·4 ± 6·6 for CD. The diagnosis of UC and CD was determined by conventional clinical, radiological, endoscopic and histological criteria as described by Lennard-Jones [27]. Patients with CD were classified according to the Vienna classification [28], briefly according to age at onset (A1: < 40 years, A2: ≥40 years), disease location (L1: terminal ileum, L2: colon, L3: ileocolon, L4: upper gastrointestinal tract) and disease behaviour (B1: non-stricturing, non-penetrating, B2: stricturing, B3: penetrating). The patients with UC were classified according to the age at onset (<40 years, ≥40 years), location of the disease (proctitis, left-sided colitis and pancolitis) and colectomy as indicator of severity. This study was carried out with the approval of the Medical Ethical Committee of the VUmc, and informed consent was obtained from all subjects.
Methods
Genomic DNA was isolated from 10 ml venous blood by a routine method. The G2964A polymorphism in the 3′ untranslated region of the STAT6 gene (NCBI SNP CLUSTER ID: rs324015) was determined by PCR and the amplification-created restriction site method according to Amoli et al. [29]. The primers used were 5′-GAA GTT CAG GCT CTG AGA GAC-3′ and 5′-CCA TCA CCC TCA GAG AGC-3′. PCR reaction was performed in the GeneAmp PCR system 9700 (PE Biosystems, CA, USA). The amplification was accomplished by an initial denaturation at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 45 s and annealing at 55°C for 45 s and extension at 72°C for 45 s, with a final extension at 72°C for 2 min. After polymerase chain reaction (PCR) amplification, electrophoresis of the reaction products was in 2% agarose gels prestained with ethidium bromide in 0·5 × Tris-borate-EDTA (TBE) buffer. Four units of BsaHI (New England Biolabs Inc., Beverly, MA, USA) were used for digestion of 7 µl PCR product at 37°C for 10 h. The digested products were visualized on 4% agarose gels stained with ethidium bromide. Individuals homozygous for the rare allele (allele 2964 A) yielded one uncut band (93 bp). The allele 2964G yielded two bands of 74 bp and 19 bp.
Statistical analysis
χ2 or Fisher's exact test were used by multiple contingency table or 2 × 2 table. When 20% of cells in the multiple contingency tables have an expected count less than 5, a necessary cell combination was performed. A value of P < 0·05 was considered statistically significant. For multiple comparisons P-values were corrected by multiplying the P-value by the number of comparisons (Bonferroni correction). Odds ratios (OR) were calculated according to Woolf's formula.
Results
The results of the genotype and allele frequencies of the STAT6 gene G2964A polymorphism of Dutch Caucasian patients with IBD and ethnically matched healthy controls are shown in Table 1. The genotypes of the healthy controls did not deviate from the expected value by Hardy–Weinberg equilibrium. No significant associations were observed in the genotype and allele frequencies between UC and CD when compared with healthy controls in Dutch Caucasians. However, a significant association between CD and healthy controls was observed in women. Here, the frequency of allele 2964 A was 0·26 in CD versus 0·18 in healthy controls, P = 0·037, OR = 1·65, 95% (CI = 1·03–2·64). However, this association did not hold after Bonferroni correction.
Table 1.
STAT6 gene G2964A genotype and allele frequencies in Dutch patients with UC, CD and healthy controls (HC)
| Genotype | Allele | |||||
|---|---|---|---|---|---|---|
| Population | No. | AAn(%) | AGn(%) | GGn(%) | AN(%) | GN(%) |
| HC | 173 | 6(3) | 57(33) | 110(64) | 69(20) | 277(80) |
| CD | 183 | 13(7) | 65(36) | 105(57) | 91(25) | 275(75) |
| UC | 141 | 6(4) | 51(36) | 84(60) | 63(22) | 219(78) |
| Male | ||||||
| HC | 79 | 4(5) | 28(35) | 47(60) | 36(23) | 122(77) |
| CD | 58 | 6(10) | 14(24) | 38(66) | 26(22) | 90(78) |
| UC | 68 | 3(4) | 31(46) | 34(50) | 37(27) | 99(73) |
| Female | ||||||
| HC | 94 | 2(2) | 29(31) | 63(67) | 33(18) | 155(82) |
| CD | 125 | 7(6) | 51(41) | 67(53) | 65(26)* | 185(74)* |
| UC | 73 | 3(4) | 20(27) | 50(69) | 26(18) | 120(82) |
n: number of individuals; N: number of alleles; percentages are given in parentheses.
: CD versus HC: Fisher's exact P = 0·0376, OR = 1·650, 95% CI = 1·031–2·641; Pc = non-significant.
As shown in Table 2 and Table 3, clinical subgroups of the patients with CD and with UC did not differ in the distribution of the STAT6 genotype and allele frequencies.
Table 2.
STAT6 gene G2964A genotype and allele frequencies in Dutch patients with CD classified according to the Vienna classification
| Genotype | Allele | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Classification | No. | AAn (%) | AGn (%) | GGn (%) | AN (%) | GN (%) | |||
| CD all | 183 | 13 (7) | 65 (36) | 105 (57) | 91 (25) | 275 (75) | |||
| Age at onset | |||||||||
| A1 | 143 | 9 (6) | 56 (39) | 78 (55) | 74 (26) | 212 (74) | |||
| A2 | 40 | 4 (10) | 9 (23) | 27 (67) | 17 (21) | 63 (79) | |||
| Location | |||||||||
| L1 | 63 | 4 (6) | 20 (32) | 39 (62) | 28 (22) | 98 (78) | |||
| L2 | 43 | 2 (5) | 16 (37) | 25 (58) | 20 (23) | 66 (77) | |||
| L3 | 74 | 6 (8) | 29 (39) | 39 (53) | 41 (28) | 107 (72) | |||
| L4 | 3 | 1 (33) | – | 2 (67) | 2 (33) | 4 (67) | |||
| Behaviour | |||||||||
| B1 | 69 | 4 (6) | 28 (40) | 37 (54) | 36 (26) | 102 (74) | |||
| B2 | 78 | 6 (8) | 25 (32) | 47 (60) | 37 (24) | 119 (76) | |||
| B3 | 36 | 3 (8) | 12 (33) | 21 (59) | 18 (25) | 54 (75) | |||
n: number of individuals; N: number of alleles; percentages are given in parentheses.
Table 3.
STAT6 gene G2964A genotype and allele frequencies in Dutch patients with UC classified according to the age, location and colectomy
| Genotype | Allele | |||||
|---|---|---|---|---|---|---|
| Classification | No. | AA n (%) | AG n (%) | GG n (%) | A N (%) | G N (%) |
| UC all | 141 | 6 (4) | 51 (36) | 84 (60) | 63 (22) | 219 (78) |
| Age at onset | ||||||
| A1 | 107 | 5 (5) | 41 (38) | 61 (57) | 51 (24) | 163 (76) |
| A2 | 34 | 1 (3) | 10 (29) | 23 (67) | 12 (18) | 56 (82) |
| Location | ||||||
| Distal colitis | 77 | 3 (4) | 31 (40) | 43 (56) | 37 (24) | 117 (76) |
| Pancolitis | 64 | 3 (5) | 20 (31) | 41 (64) | 26 (20) | 102 (80) |
| Colectomy | ||||||
| Yes | 46 | 3 (6) | 17 (37) | 26 (57) | 23 (25) | 69 (75) |
| No | 95 | 3 (3) | 34 (36) | 58 (61) | 40 (21) | 150 (79) |
n: number of individuals;
N: number of alleles; percentages are given in parentheses.
Discussion
In the present study, we found no significant association of the G2964A polymorphism in the STAT6 gene with Dutch Caucasian patients with IBD. The frequency of allele 2964 A was slightly increased in female CD patients when compared with female healthy controls. As shown in this study the 2964 A allele is the less frequent allele in the Dutch Caucasian population. This has also been found previously in a UK Caucasian population [24,29]. In contrast, Gao et al. showed the 2964G allele to be the minor allele in Japanese subjects [24]. Furthermore, we did not find an association of this polymorphism with subgroups of CD patients clinically classified according to the Vienna classification, and with subgroups of UC classified with age, disease location and colectomy performed. Our results have shown that the STAT6 gene G2964A polymorphism does not predispose to CD or UC in Dutch Caucasian patients and does not influence the course of the disease.
Recent genome-wide linkage studies have shown that IBD2 susceptibility locus is mapped on chromosome 12q [10–14]. The STAT6 gene is located well within this region. Since STAT6 is a component involved in the IL-4 signalling pathway, it is interesting to know that the IL-4RA gene is located in chromosome 16p12 within the IBD1 locus and the IL-4 gene has been mapped to the IBD5 susceptibility region for CD on chromosome 5q31–33 [30]. Even though the IL-4RA gene polymorphism 576R alone [31] has been found not to be associated with IBD, the combination of IL-4RA gene Q576R and IL-4–34T alleles were found to be a risk factor for CD in a study from the UK [32]. A German study did also show evidence for a weak association between the IL-4–590T gene polymorphism and CD [33]. The study of Rioux et al. [30] showed an association between CD and a specific common haplotype of the cytokine region in 5q31 including the IL-4 gene. In the Dutch population, genotypes for both the IL-4RA S478P (in strong linkage disequilibrium with Q576R) and a −1111 promoter SNP in the IL-13 gene revealed synergy in asthma susceptibility [34].
In this study, we found no significant association between the STAT6 G2964A gene polymorphism and IBD in Dutch Caucasians. Significant interaction between genes in the same pathway is not unprecedented. Further studies will focus on other polymorphisms located in regulatory and coding regions and at exon/intron boundaries of the STAT6 gene on chromosome 12q for susceptibility to IBD and explore interactions with IL-4RA and IL-13, particularly in relation to UC that has features compatible with Th2 dysregulation as opposed to CD where a Th1 response predominates.
Acknowledgments
This study was performed in the Laboratory of Immunogenetics of the Vrije Universiteit Medical Centre Amsterdam. The study was supported by grants of the Natural Science Foundation of China (30070350), and the Royal Netherlands Academy of Art and Sciences (KNAW 00CDP024) to Prof. B. Xia and Prof. A.S. Peña. Dr J. Wu received a scholarship from the Chinese Scholarship Council.
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