Polymorphisms of STAT4 and the risk of inflammatory bowel disease: A case-control study in Chinese Han population
- Authors:
- Published online on: January 21, 2013 https://doi.org/10.3892/br.2013.59
- Pages: 320-324
Abstract
Introduction
Inflammatory bowel disease (IBD) is a series of inflammatory conditions of the colon and small intestine comprising Crohn’s disease (CD) and ulcerative colitis (UC) (1–4). IBD, with a complex pathogenesis influenced by a combination of genetic risk factors and environmental events, is highly heritable, thus investigations into the genetic susceptibility of this disease are ongoing (5,6). Numerous loci, such as NOD2, IL23R, ATG16L1 and IRGM, are associated with IBD susceptibility (7–14). However, the genetic polymorphisms located within these genes are not sufficient to adequately explain the pathogenesis and development of IBD and its various phenotypes.
Signal transducer and activator of transcription 4 (STAT4), a transcription factor whose gene is located on chromosome 2q33, is important in the development of inflammation of various immune-mediated diseases (15). STAT4 variation has been reported to be significantly associated with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) (16). The association of STAT4 haplotype characterized by the rs7574865 polymorphism with IBD was originally reported by Martinez et al (17) in a Spanish population, a study that was replicated by Diaz-Gallo et al (18). Glas et al (19) reported that rs7574865 polymorphism was associated with colonic CD and early disease onset in Caucasians.
These associations need to be confirmed by replication studies, particularly in other ethnic populations. Additionally, the differences in risk allele frequencies and linkage disequilibrium structure in ethnicities may provide insights to refine the association signal and identify the true risk variant. Therefore, the aim of the present study was to evaluate whether the previously identified STAT4 single-nucleotide polymorphisms (SNPs) are associated with IBD susceptibility in a population-based Chinese Han cohort including 562 unrelated individuals (232 CD, 56 UC and 274 healthy controls).
Patients and methods
Human subjects
A total of 288 unrelated Chinese patients with IBD (232 with CD and 56 with UC) and 274 matched healthy controls were included in this study. The patients were recruited from the Department of Gastroenterology of the Ruijin Hospital Affiliated to the Shanghai Jiaotong University School of Medicine. The patients were diagnosed by senior physicians according to standard clinical, endoscopic, radiologic and histological criteria (20,21). Healthy individuals with no history of digestive system disease, unrelated to each other or to the patients were randomly selected under routine health screening as controls (Table I). The study was approved by the Research Ethics Committee of the Ruijin Hospital and the enrolled patients provided informed consent to participate in the study.
STAT4 genotyping
Genomic DNA was extracted from whole blood samples using QIAamp blood extraction kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions. The DNA samples were genotyped for the rs7574865 and rs7582694 polymorphisms via polymerase chain reaction (PCR) with sequence-specific primers. The primer sequences, designed using the genomic sequences in the GenBank (http://www.ncbi.nlm.nih.gov) are listed in Table II. Genotyping of the amplified products was assessed for the presence/absence of PCR amplicons specific to the particular alleles by using a standard 2% agarose gel electrophoresis with ethidium bromide staining. Then, ∼10% samples were confirmed using Sanger sequencing.
Statistical analysis
Hardy-Weinberg equilibrium testing (HWE), P-value computations (P>0.05), in the healthy control and patient groups, calculations of allelic and genotypic associations of SNPs with susceptibility to IBD were performed using the SHEsis software (http://analysis.bio-x.cn) (22,23). The tests were two-tailed and P<0.05 was considered to indicate a statistically significant difference.
Results
Patients
To evaluate the association of the STAT4 polymorphisms with IBD in the Chinese Han population, we genotyped SNP rs7574865 and rs7582694 among 232 CD and 56 UC patients, as well as 274 healthy controls. Characteristics of the study subjects are shown in Table I.
Genotypic and allele frequencies
Distributions of the genotype and allele frequencies of the two SNPs between the cases and healthy controls are shown in Table III. Results revealed that the STAT4 rs7574865 genotype was significantly different between CD/IBD patients and the controls. However, the association analysis showed that neither of the two SNPs was significantly associated with IBD susceptibility (rs7574865 CD: P=0.66, OR=0.95, 95% CI 0.74–1.21; UC: P=0.43, OR=0.85, 95% CI 0.56–1.28; IBD: P=0.52, OR=0.93, 95% CI 0.73–1.17; rs7582694 CD: P=0.40, OR=1.12, 95% CI 0.86–1.44; UC: P=0.50, OR=0.86, 95% CI 0.56–1.33; IBD: P=0.62, OR=1.06, 95% CI 0.83–1.36) (Table III).
Discussion
IBD is a series of inflammatory conditions of the colon and small intestine, characterized by a combination of genetic risk factors and environmental events. Numerous gene variants are reportedly associated with IBD or IBD-related phenotypes (7–14). However, these variants are not sufficient to adequately explain the pathogenesis and development of IBD and its various phenotypes. The STAT4 gene has been previously reported to be significantly associated with certain autoimmune diseases such as SLE, RA (16,24,25) and psoriasis (24,26).
The Chinese Han is one of the largest populations in the world. Evaluation of the association of risk factors with specific diseases is crucial. In this study, we present a genotype-phenotype analysis of STAT4 gene polymorphisms in an IBD cohort. This study did not reproduce the association of STAT4 variants with susceptibility to IBD in the Chinese population. However, we found the genotype frequency of rs7574865 to vary significantly in CD/IBD patients and the healthy controls. Previous studies have shown that the STAT4 SNP rs7574865 was significantly associated with RA (16), IBD and type I diabetes (17). Additionally, the association analysis of rs7574865 with IBD was carried out independently by several groups based on various ethnic populations. Unlike the original findings of Martinez et al (17), Glas et al (19), who analyzed 2,704 individuals of Caucasian origin, hypothesized that rs7574865 was associated with CD and early disease onset, but not with UC. Nevertheless, in another independent Spanish cohort Diaz-Gallo et al (18) observed that rs7574865 was associated with UC but not CD. A further discrepancy was noted by Moon et al (27) who analyzed eight STAT4 SNPs in 657 unrelated Korean participants, and did not detect any statistically significant association between rs7574865 and CD or UC. These varying results may be due to the variation of complex circumstances and extended genetic background.
Other SNPs of STAT4, such as rs7582694, also analyzed in our study, may affect several autoimmune diseases in various ethic populations (28–30). However, rs7582694 was not found to be associated with IBD. Although numerous gene variants are reportedly associated with IBD susceptibility (7,10–14), a re-evaluation of the association is required in additional ethic populations. Lack of replication has long been a challenge in genetic association studies of IBD.
Taken together, although our study was sufficiently powered, there was no evidence to show that the previously reported common variants rs7574865 and rs7582694 in the STAT4 gene increased the risk of IBD in a Chinese Han population. However, this study has a limitation. Although the two SNPs were not associated with IBD susceptibility and phenotypes, the relatively small study population caused the weak diversity of the genotype frequency of rs7574865 in the CD patients and controls. The association analysis in our study should be replicated in a larger sample-sized study of Chinese patients, as well as in other ethnic populations.
Acknowledgements
This study was funded by the Science and Technology Commission of the Municipality of Shanghai (grant no. 10JC1410300 to Z.G. Wang) and the National Natural Science Foundation of China (grant no. 31000408 to H.X. Zhang and 81201365 to W.Y. Xu). The authors would like to thank the members of the cohort for their devoted contribution to a scientific discovery.
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