Abstract
Aims: The aim of our study was to evaluate the association between CTLA-4 polymorphisms (+49A/G, -318C/T and CT60A/G) and ankylosing spondylitis (AS) susceptibility. Methods: A total of 120 AS cases and healthy controls, matched on the age and gender, were enrolled in the study. Polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) were used to determine the gentypes of +49A/G, -318C/T and CT60A/G polymorphisms. Genotype distribution in control group was assessed by Hardy Weinberg Equilibrium (HWE) test. Odds ratio (OR) with 95% confidence interval (95% CI) were adopted to evaluate the relationship of CTLA-4 polymorphisms and AS susceptibility. Results: In our study, genotype distribution of the three polymorphisms in control group was consistent with the HWE (P > 0.05). The genotype analysis showed that AA genotype of + 49A/G polymorphism could increase the risk for AS (OR=2.357, 95% CI=1.127-4.930). Moreover, the frequency of A allele was also presented as a risk factor for AS. Additionally, AA genotype and A allele of CT60A/G appeared to be related with AS susceptibility (OR=2.610, 95% CI=1.047-6.510; OR=1.751, 95% CI=1.160-2.641). However, the T allele of -318C/T appeared to be a protective factor for AS (OR=0.383, 95% CI=0.228-0.643). Conclusion: In summary, there existed significant association between CTLA-4 gene polymorphisms and increased or decreased risk for AS.
Keywords: CTLA-4, ankylosing spondylitis, +49A/G, -318C/T, CT60A/G
Introduction
Ankylosing spondylitis (AS) is one of the most prevalent prototypical spondyloarthropathies. The disease is more prevalent in Whites population, morbidity of which is 0.1-1%, higher than other population [1-3]. Moreover, it has been demonstrated that the occurrence of AS is highest in northern European countries and lowest in sub-Saharan Africa [4,5]. The probability to develop AS for individuals who are positive for HLA-B27 is approximately 1-2% and will up to 15-20% when they have a first-degree relative with AS [6,7]. Although some treatments including medication, surgery and physical therapy have been used to relieve symptoms, there is still no effective therapy for AS [8,9]. Therefore, it is urgent to find out high-risk population of AS and prevent the disease timely. Recently, it has been reported that specific cytotoxic T-lymphocyte antigen 4 (CTLA-4) gene polymorphisms may play crucial role in the pathogenesis of AS.
CTLA-4, also known as cluster of differentiation 152 (CD152), is a negative regulator of T-cell immune response and is expressed primarily in activated regulatory T-cells [10-12]. Stimulating the CTLA-4 receptor can turn off the attack of T cells, which are a type of lymphocyte (a type of white blood cell). Numerous autoimmune diseases have been reported to be associated with polymorphisms of CTLA-4 gene. Polymorphisms within CTLA-4 that was related with the down-regulation of CTLA-4 could cause autoimmune T cell clonal proliferation and thus result in the occurrence of autoimmune diseases.
Until now, the studies have investigated the relationship of CTLA-4 +49A/G polymorphism and primary biliary cirrhosis, 318C/T polymorphism and systemic sclerosis, and CT60A/G polymorphism and autoimmune thyroid disease [13-15]. However, there were few studies on the association of above polymorphisms and AS susceptibility.
The aim of our study was to assess the association of CTLA-4 + 49A/G polymorphism, 318C/T polymorphism and CT60A/G polymorphism with AS risk.
Subjects and analysis
Subjects
The peripheral blood of AS patients was collected from department of spinal surgery, provincial hospital affiliated to Shandong University, and healthy controls were selected from blood donors. The AS group includes 120 patients with 54 men and 66 women and the average age were 22.4 years and 49.6 years, respectively. One hundred and twenty healthy people, 58 men and 62 women, who had never suffered from any rheumatological disease, were selected as the control group. Their average age was 26.7 and 47.7. Controls were matched with AS cases on the age and gender [16]. The subjects would be excluded if they were met the following items: high blood pressure, diabetes, hyperlipidemia, smoking and obesity. Written informed consent was obtained from all subjects. The experiment was supported by the Hospital’s Ethics Committee.
DNA extraction and genotyping analysis
Genome DNA was isolated from peripheral blood samples by the conventional phenol-chloroform extraction method. CTLA-4 polymorphisms were examined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing analyses. Primers were designed by Primer 5.0 software, provided by Shanghai biological engineering technology service co., LTD. Primers sequences were listed in Table 1.
Table 1.
Primers sequences of CTLA-4 polymorphisms
| SNP locus | SNP site | SNP | Sequence |
|---|---|---|---|
| +49A/G | Exon1 | rs231775 | F: 5’-CCACGGCTTCCTTTCTCGTA-3’ |
| R: 5’-AGTCTCACTCACCTTTGCAG-3’ | |||
| -318C/T | Promoter | rs5742909 | F: 5’-AAATGAATTGGACTGGATGGT-3’ |
| R: 5’-TTACGAGAAAGGAGGCCGTG-3’ | |||
| CT60A/G | 3’-flanking region | rs3087243 | F: 5’-CACCACTATTTGGGATAT ACC-3’ |
| R: 5’-AGGTCTTATTTTGAGGAAGGC-3’ |
20 μL reaction solution contained 10 × PCR buffer 2.5 μL, MgCl2 (25 mmol/L) 1.5 μL, dNTPs (2.5 mmol/L of each) 2.0 μL, each primer (20/μmol/L) 0.4 μL, DNA template 1.5 μL, Taq polymerase (5 U/μL0.2 μL (Nanjing Dulai Biol Technol Co., LTD), and double-distilled water 16.5 μL. Amplifications were performed in 35 cycles of 30 s at 94°C, 30 s at 60°C, 30 s at 72°C, 20 s at 95°C, 30 s at 58.7°C, 58.6°C, 58.5°C, 30 s extension at 72°C and a final 5 min extension at 72°C + 49A/G and CT60A/G polymorphism were genotyped by PCR-RFLP. The PCR products were digested by BbvI, and NcoI restriction enzyme (Boehringe, Germany). And the digestion was performed at 37.5°C for BbvI and 65°C for NcoI overnight. All digested products were detected by 20 g/L agarose gel electrophoresis with 80 v for 30 min, then stained with ethidium bromide and analyzed by gel imaging (GeneScan3.1 software, PE Biosystems). And the genotypes of -318C/T were determined by direct sequencing.
Statistical analysis
Odds ratio (OR) and 95% confidence interval (CI), calculated with X2 test, were employed to analyze the association of genotype or allele and AS susceptibility. For control group, the observed genotype frequencies of the three polymorphisms were assessed with Hardy-Weinberg equilibrium (HWE) by Pearson X2 test. P value less than 0.05 was considered as significant. All the analysis was conducted in SPSS 18.0 software.
Results
The electrophoresis results of enzyme digestion and sequence for three polymorphisms of CTLA-4
For +49A/G, the digested results showed that homozygous AA was corresponded with 328 bp fragment, heterozygous AG with 328 bp, 244 bp and 84 bp fragments and homozygous GG with 244 bp and 84 bp fragments. For CT60A/G, we found that homozygous GG genotype were presented with 216 bp bands, heterozygous AG individuals with 26 bp, 216 bp and 196 bp bands and homozygous AA with 26 bp and 196 bp bands. In addition, -318C/T polymorphism produced three genotypes of CC, CT and TT.
Relationship of CTLA-4 polymorphisms (+49A/G, 318C/T and CT60A/G) with risk for AS
As shown in Table 2, Genotype distribution of the three polymorphisms in control group were consistent with the HWE (P>0.05). And our study showed that AA genotype of +49A/G polymorphism could increase the risk for AS (OR=2.357, 95% CI=1.127-4.930). Moreover, the frequency of A allele was higher in case group (20.8% vs. 40.0%), which was presented as a risk factor for AS. Additionally, AA genotype of CT60A/G appeared to be related with AS susceptibility (OR=2.610, 95% CI=1.047-6.510). Further analysis suggested that A allele of CT60A/G also showed strong effects on the occurrence of AS (OR=1.751, 95% CI=1.160-2.641). However, the T allele of -318C/T could inhibit the occurrence of AS (OR=0.383, 95% CI=0.228-0.643).
Table 2.
Alleles and genotypes distribution of CTLA-4 polymorphisms in case and control groups
| Genotype/Allele | Case (n=120), n (%) | Control (n=120), n (%) | χ2 | P value | OR (95% CI) |
|---|---|---|---|---|---|
| Genotype | |||||
| 49A/G | |||||
| GG | 28 (23.3) | 44 (36.7) | - | - | 1 |
| AG | 62 (51.7) | 56 (46.7) | 3.344 | 0.067 | 1.740 (0.959-3.157) |
| AA | 30 (25.0) | 20 (16.6) | 5.273 | 0.022 | 2.357 (1.127-4.930) |
| 318C/T | |||||
| CC | 98 (81.7) | 70 (58.3) | - | - | 1 |
| CT | 20 (16.7) | 46 (38.3) | 14.893 | 0.000 | 0.311 (0.169-0.570) |
| TT | 2 (1.6) | 4 (3.4) | 1.481 | 0.224 | 0.357 (0.064-2.004) |
| CT60A/G | |||||
| AA | 77 (64.2) | 59 (49.2) | 4.441 | 0.035 | 2.610 (1.047-6.510) |
| AG | 35 (29.2) | 45 (37.5) | 0.826 | 0.363 | 1.556 (0.598-4.050) |
| GG | 8 (6.6) | 16 (13.3) | - | - | 1 |
| Allele | |||||
| 49A/G | |||||
| G | 118 (49.2) | 144 (60.0) | - | - | 1 |
| A | 122 (50.8) | 96 (40.0) | 5.681 | 0.017 | 1.551 (1.080-2.226) |
| 318C/T | |||||
| C | 216(90.0) | 186(77.5) | - | - | 1 |
| T | 24(10.0) | 54(22.5) | 13.777 | 0.000 | 0.383(0.228-0.643) |
| CT60A/G | |||||
| G | 51 (21.3) | 77 (32.1) | - | - | 1 |
| A | 189 (78.7) | 163 (67.9) | 7.202 | 0.007 | 1.751 (1.160-2.641) |
Discussion
Common genetics (human leukocyte antigen [HLA] class-I gene, HLA-B27) and common pathology (enthesitis) are related to the spondyloarthropathies. AS is the main disease linked with HLA gene (1973) and the direct relationship of AS with HLA-B27 gene has been determined [17-20], which suggests that genes play crucial role in the pathogenesis of AS.
The effects of CTLA-4 +49A/G, -318C/T and CT60A/G polymorphisms on other diseases have been extensively reported [21-25]. The study of CHEN et al. indicated that there existed an obvious association between PBC and CTLA-4 +49A/G polymorphism [13]. For Italian population, a relationship between -318C/T polymorphism with risk of systemic sclerosis has been found in the study of Balbi et al [14]. Moreover, according to Bicek et al’s study, CT60A/G polymorphism was related with GD susceptibility, but not with two other types of autoimmune thyroid disease (AITD) (HT and PPT) [15]. In view of the above-mentioned studies, we concluded that CTLA-4 polymorphisms might serve as risk factor for AS, so the relationship of CTLA-4 and AS risk was investigated. Our study showed that + 49A/G and CT60A/G polymorphisms were both genetic-susceptibility factors for AS. While, -318C/T polymorphism was related with decreased risk for AS.
The fact is that the studies about the association of gene polymorphism and disease risk commonly focus on the single SNP. To explore the precise relationship of CTLA-4 polymorphism, we studied three SNPs located in exon1, promoter and 3’-flanking region of CTLA-4, which makes our results much more comprehensive and accuracy. In conclusion, there was significant correlation between CTLA-4 polymorphisms and AS susceptibility. Further studies with consideration of gene-gene, gene-environment interaction are needed to confirm the results.
Disclosure of conflict of interest
None.
References
- 1.Braun J, Bollow M, Remlinger G, Eggens U, Rudwaleit M, Distler A, Sieper J. Prevalence of spondylarthropathies in HLA-B27 positive and negative blood donors. Arthritis Rheum. 1998;41:58–67. doi: 10.1002/1529-0131(199801)41:1<58::AID-ART8>3.0.CO;2-G. [DOI] [PubMed] [Google Scholar]
- 2.Trontzas P, Andrianakos A, Miyakis S, Pantelidou K, Vafiadou E, Garantziotou V, Voudouris C. Seronegative spondyloarthropathies in Greece: a population-based study of prevalence, clinical pattern, and management. The ESORDIG study. Clin Rheumatol. 2005;24:583–589. doi: 10.1007/s10067-005-1106-9. [DOI] [PubMed] [Google Scholar]
- 3.De Angelis R, Salaffi F, Grassi W. Prevalence of spondyloarthropathies in an Italian population sample: a regional community-based study. Scand J Rheumatol. 2007;36:14–21. doi: 10.1080/03009740600904243. [DOI] [PubMed] [Google Scholar]
- 4.Reveille JD, Ball EJ, Khan MA. HLA-B27 and genetic predisposing factors in spondyloarthropathies. Curr Opin Rheumatol. 2001;13:265–272. doi: 10.1097/00002281-200107000-00004. [DOI] [PubMed] [Google Scholar]
- 5.Taurog JD. The mystery of HLA-B27: if it isn’t one thing, it’s another. Arthritis Rheum. 2007;56:2478–2481. doi: 10.1002/art.22807. [DOI] [PubMed] [Google Scholar]
- 6.van der Linden S, van der Heijde D. Ankylosing spondylitis. Clinical features. Rheum Dis Clin North Am. 1998;24:663–676. vii. doi: 10.1016/s0889-857x(05)70036-3. [DOI] [PubMed] [Google Scholar]
- 7.Khan MA. Update on spondyloarthropathies. Ann Intern Med. 2002;136:896–907. doi: 10.7326/0003-4819-136-12-200206180-00011. [DOI] [PubMed] [Google Scholar]
- 8.Toivanen A, Mottonen T. Ankylosing spondylitis: current approaches to treatment. BioDrugs. 1998;10:193–200. doi: 10.2165/00063030-199810030-00003. [DOI] [PubMed] [Google Scholar]
- 9.Williams RO, Paleolog E, Feldmann M. Cytokine inhibitors in rheumatoid arthritis and other autoimmune diseases. Curr Opin Pharmacol. 2007;7:412–417. doi: 10.1016/j.coph.2007.06.001. [DOI] [PubMed] [Google Scholar]
- 10.Ueda H, Howson JM, Esposito L, Heward J, Snook H, Chamberlain G, Rainbow DB, Hunter KM, Smith AN, Di Genova G, Herr MH, Dahlman I, Payne F, Smyth D, Lowe C, Twells RC, Howlett S, Healy B, Nutland S, Rance HE, Everett V, Smink LJ, Lam AC, Cordell HJ, Walker NM, Bordin C, Hulme J, Motzo C, Cucca F, Hess JF, Metzker ML, Rogers J, Gregory S, Allahabadia A, Nithiyananthan R, Tuomilehto-Wolf E, Tuomilehto J, Bingley P, Gillespie KM, Undlien DE, Ronningen KS, Guja C, Ionescu-Tirgoviste C, Savage DA, Maxwell AP, Carson DJ, Patterson CC, Franklyn JA, Clayton DG, Peterson LB, Wicker LS, Todd JA, Gough SC. Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease. Nature. 2003;423:506–511. doi: 10.1038/nature01621. [DOI] [PubMed] [Google Scholar]
- 11.Thompson CB, Allison JP. The emerging role of CTLA-4 as an immune attenuator. Immunity. 1997;7:445–450. doi: 10.1016/s1074-7613(00)80366-0. [DOI] [PubMed] [Google Scholar]
- 12.Kristiansen OP, Larsen ZM, Pociot F. CTLA-4 in autoimmune diseases--a general susceptibility gene to autoimmunity? Genes Immun. 2000;1:170–184. doi: 10.1038/sj.gene.6363655. [DOI] [PubMed] [Google Scholar]
- 13.Bicek A, Zaletel K, Gaberscek S, Pirnat E, Krhin B, Stopar TG, Hojker S. 49A/G and CT60 polymorphisms of the cytotoxic T-lymphocyte-associated antigen 4 gene associated with autoimmune thyroid disease. Hum Immunol. 2009;70:820–824. doi: 10.1016/j.humimm.2009.06.016. [DOI] [PubMed] [Google Scholar]
- 14.Balbi G, Ferrera F, Rizzi M, Piccioli P, Morabito A, Cardamone L, Ghio M, Palmisano GL, Carrara P, Pedemonte S, Sessarego M, De Angioletti M, Notaro R, Indiveri F, Pistillo MP. Association of -318C/T and +49A/G cytotoxic T lymphocyte antigen-4 (CTLA-4) gene polymorphisms with a clinical subset of Italian patients with systemic sclerosis. Clin Exp Immunol. 2007;149:40–47. doi: 10.1111/j.1365-2249.2007.03394.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Chen RR, Han ZY, Li JG, Shi YQ, Zhou XM, Wang JB, Cai XQ, Wang XC, Han Y, Fan DM. Cytotoxic T-lymphocyte antigen 4 gene +49A/G polymorphism significantly associated with susceptibility to primary biliary cirrhosis: a meta-analysis. J Dig Dis. 2011;12:428–435. doi: 10.1111/j.1751-2980.2011.00537.x. [DOI] [PubMed] [Google Scholar]
- 16.van der Linden S, Valkenburg HA, Cats A. Evaluation of diagnostic criteria for ankylosing spondylitis. A proposal for modification of the New York criteria. Arthritis Rheum. 1984;27:361–368. doi: 10.1002/art.1780270401. [DOI] [PubMed] [Google Scholar]
- 17.Schlosstein L, Terasaki PI, Bluestone R, Pearson CM. High association of an HL-A antigen, W27, with ankylosing spondylitis. N Engl J Med. 1973;288:704–706. doi: 10.1056/NEJM197304052881403. [DOI] [PubMed] [Google Scholar]
- 18.Brewerton DA, Hart FD, Nicholls A, Caffrey M, James DC, Sturrock RD. Ankylosing spondylitis and HL-A 27. Lancet. 1973;1:904–907. doi: 10.1016/s0140-6736(73)91360-3. [DOI] [PubMed] [Google Scholar]
- 19.Caffrey MF, James DC. Human lymphocyte antigen association in ankylosing spondylitis. Nature. 1973;242:121. doi: 10.1038/242121a0. [DOI] [PubMed] [Google Scholar]
- 20.Alvarez I, Lopez de Castro JA. HLA-B27 and immunogenetics of spondyloarthropathies. Curr Opin Rheumatol. 2000;12:248–253. doi: 10.1097/00002281-200007000-00003. [DOI] [PubMed] [Google Scholar]
- 21.Shojaa M, Aghaie M, Amoli M, Javid N, Shakeri F, Khashayar P, Tabarraei A, Keshtkar AA, Joshaghani HR, Kouroshnia A, Qorbani M, Mahmoudi F, Mohebbi R, Ranjbarpour N. Association between 318C/T polymorphism of the CTLA-4 gene and systemic lupus erythematosus in Iranian patients. Int J Rheum Dis. 2014 doi: 10.1111/1756-185X.12275. [Epub ahead of print] [DOI] [PubMed] [Google Scholar]
- 22.Jaiswal PK, Singh V, Mittal RD. Cytotoxic T lymphocyte antigen 4 (CTLA4) gene polymorphism with bladder cancer risk in North Indian population. Mol Biol Rep. 2014;41:799–807. doi: 10.1007/s11033-013-2919-2. [DOI] [PubMed] [Google Scholar]
- 23.Ghapanchi J, Haghshenas MR, Ghaderi H, Amanpour S, Nemati V, Kamali F. Ctla-4 gene polymorphism in +49a/g position: a case control study on patients with oral lichen planus. J Int Oral Health. 2014;6:17–21. [PMC free article] [PubMed] [Google Scholar]
- 24.Liu J, Zhang HX. Association between the Rs3087243 polymorphism and risk for diabetes: a meta-analysis. Genet Mol Res. 2013;12:6344–6350. doi: 10.4238/2013.December.6.1. [DOI] [PubMed] [Google Scholar]
- 25.Pastuszak-Lewandoska D, Domanska D, Rudzinska M, Bossowski A, Kucharska A, Sewerynek E, Czarnecka K, Migdalska-Sek M, Czarnocka B. CTLA-4 polymorphisms (+49A/G and -318C/T) are important genetic determinants of AITD susceptibility and predisposition to high levels of thyroid autoantibodies in Polish children - preliminary study. Acta Biochim Pol. 2013;60:641–646. [PubMed] [Google Scholar]