Archives of Medical Research 42 (2011) 235e238

ORIGINAL ARTICLE

Association between CTLA-4 Exon-1 þ49AOG Polymorphismand Primary Biliary Cirrhosis Risk: A Meta-analysis

Qiang Huang,a,* Feng Shao,a,* Cheng Wang,a Lu-jun Qiu,a Yuan-guo Hu,a and Ji-hai Yub

aDepartment of Hepato-Biliary Surgery, Anhui Provincial Hospital Affiliated with Anhui Medical University, Anhui, ChinabAnhui Medical University, Anhui, China

Received for publication January 23, 2011; accepted March 25, 2011 (ARCMED-D-11-00043).

*These authors con

Address reprint r

Biliary Surgery, Anhu

University, 17 Lujiang

and Fax: þ86-551-22

0188-4409/$ - see frodoi: 10.1016/j.arcm

Background and Aims. CTLA-4 exon-1 þ49AOG polymorphisms have been reported toinfluence the risk for primary biliary cirrhosis in many studies; however, the results stillremain controversial and ambiguous. The aim of this study was to determine more preciseestimations for the relationship between CTLA-4 exon-1 þ49AOG polymorphisms andthe risk for primary biliary cirrhosis.

Methods. Electronic searches for all publications were conducted on associationsbetween this variant and breast cancer in several databases through November 2010.Crude odds ratios (ORs) with 95% confidence intervals (CIs) were calculated to estimatethe strength of the association. Eight studies were identified including 2151 cases and2214 controls.

Results. Overall, there were no significant associations between CTLA þ49GOA poly-morphism and primary biliary cirrhosis risk (codominant model: GA vs. AA OR 51.190, 95% CI 5 0.818e1.732; GG vs. AA OR 5 1.153, 95% CI 5 0.858e1.550; domi-nant model: OR5 1.181, 95% CI5 0.873e1.599; and recessive model: OR5 1.148; 95%CI 5 0.903e1.459). In the subgroup analysis by ethnicity, a significantly increased riskwas found for Asians (GG vs. AA OR 5 1.873; 95% CI 5 1.202e2.921) and recessivemodel (OR5 1.758; 95% CI5 1.271e2.433). In the stratified analysis by control sources,significant association were observed in population-based studies (GAvs. AAOR5 1.432;95% CI 5 1.078e1.902).

Conclusions. This meta-analysis suggests that the CTLA-4 þ49GOA polymorphismmay be a risk factor for primary biliary cirrhosis in Asians. � 2011 IMSS. Publishedby Elsevier Inc.

Key Words: Cytotoxic T-lymphocyte antigen 4, Polymorphism, Primary biliary cirrhosis,

Meta-analysis.

Introduction

Primary biliary cirrhosis (PBC) is a chronic cholestatic liverdisorder characterized by immune-mediated damage to thebiliary epithelial cells lining the small intrahepatic bile ducts(1). Multiple genetic and environmental factors are consid-ered to be involved in the pathogenesis of PBC (2e4). Anassociation between human leukocyte antigen (HLA-DRB)

tributed equally to this work.

equests to: Qiang Huang, Department of Hepato-

i Provincial Hospital Affiliated with Anhui Medical

Road, Hefei 230001, Anhui Province, China; Phone

83325; E-mail: tagsmile1985@163.com

nt matter. Copyright � 2011 IMSS. Published by Elseviered.2011.04.004

and genetic susceptibility to PBC has been widely docu-mented for many populations (5e10). However, HLA hasbeen estimated to account for |15% of the genetic compo-nent of susceptibility to PBC, so several other genes maybe involved in the pathogenesis of PBC (11e12).

In order to have a better understanding about the etiologyof PBC, genes other than HLA must be explored. In recentyears, several nonmajor histocompatibility complex immuno-regulatory genes have been identified in PBC. An importantgene is the T-cell regulatory gene cytotoxic T-lymphocyte-associated antigen-4 (CTLA4) located on chromosome2q33 in humans and comprised of four exons and three introns(13). A common single nucleotide polymorphism at position

Inc.

236 Huang et al./ Archives of Medical Research 42 (2011) 235e238

49 of exon 1 was described by Nistico et al., which causes anamino acid change (threonine to alanine) in the peptide leadersequence of the CTLA-4 protein (14).

To date, many epidemiological studies have been doneto evaluate the association between CTLA þ49GOA poly-morphism and PBC (15e22). However, the results wererather inconsistent, partially due to the relative smallsample size of individual studies. Therefore, we performedthis meta-analysis of eight eligible studies to get a moreprecise estimation of the association.

Materials and Methods

Publication Search

Articles were identified by an electronic search on Medline,Embase and the Cochrane Library using the followingkeywords and subject terms: cytotoxic T lymphocyte-associated antigen 4, CTLA-4, polymorphism, primarybiliary cirrhosis and PBC (last search: November 30,2010). Eligible studies were retrieved and examined care-fully. Review articles and bibliographies of other relevantstudies identified were hand-searched to find additionaleligible studies. When more than one of the same populationwas included in several articles, only the most recent or withcomplete study was included in this meta-analysis. Includedstudies had to fulfill the following criteria: 1) evaluation ofthe CTLA-4 exon-1 þ49A/G polymorphism and PBC risk;2) caseecontrol study; and 3) at least two comparison groups(PBC group vs. control group).

Data Extraction

Two authors (FS and JHY) independently extracted dataand entered them in a customised database. Discrepanciesabout inclusion of studies and interpretation of data wereresolved by discussion, consensus and arbitration by QLJor QH. The following data were collected from each study:

Table 1. Characteristics of studies included in this meta-analysis

Author Year Country Origin Source of control

Donaldson 2007 Italy C Population

Donaldson 2007 UK C Population

Schott 2007 Germany M Population

Fan 2004 China Asian Hospital

Oertelt 2005 Italy C Hospital

Bittencourt 2003 Brazil M Population

Agarwal 2000 UK C Population

Juran 2010 Canada C Hospital

Juran 2010 USA C Hospital

Joshita 2010 Japan A Hospital

Joshita 2010 Japan A Hospital

C, Caucasian; M, mixed; A, Asian; HWE, HardyeWeinberg equilibrium.

first author’s name, year of publication, ethnicity, countryof origin, sources of controls, genotyping method, HardyeWeinberg equilibrium (HWE) and number of differentgenotypes in cases and controls.

Statistical Methods

Crude ORs with 95% CIs were used to assess the strength ofassociation between the CTLA þ49GOA polymorphismand PBC risk.We evaluated the risk of the codominantmodel(GG vs. AA; GA vs. AA), the dominant model (GG/GA vs.AA) and the recessive model (GG vs.GA/AA), respectively.Heterogeneity assumption was checked by the c2 basedQ-test and was regarded to indicate significance for p !0.1(23). The fixed model would be used if the test of heteroge-neity was not significant; otherwise, the random-effectmodel would be used (24,25). Heterogeneity among studieswas assessed by I2 statistic, which was interpreted as theproportion of total variation contributed among study vari-ants. Sensitivity analysis was carried out by including andexcluding studies not in HWE (26). An estimate of potentialpublication bias was evaluated by the funnel plot inwhich thestandard error of log (OR) of each study was plotted againstits log (OR). An asymmetric plot suggested a possible publi-cation bias. Funnel plot asymmetry was assessed usingEgger’s linear regression test. If p !0.05, publication biaswas statistically significant. Subgroup analyses wereperformed by ethnicities and sources of controls. All statis-tical analyses were carried out with STATA software,v.10.0 (STATA Corp., College Station, TX).

Results

Study Characteristics

A total of eight studies fulfilling the inclusion criteria wereidentified. In three of these publications, ORs were presented

Genotype distribution

HWE c2 (P)

Case Control

AA AG GG AA AG GG

40 29 4 54 35 10 1.399 (0.237)

75 130 38 106 142 43 0.167 (0.683)

58 90 32 78 87 37 2.085 (0.149)

6 34 37 23 93 44 5.351 (0.021)

27 87 40 45 72 49 2.894 (0.089)

23 23 4 29 30 8 0.003 (0.955)

57 106 37 99 80 21 0.638 (0.424)

58 142 160 43 179 182 0.011 (0.918)

83 215 208 36 153 168 0.018 (0.893)

25 90 83 29 83 58 0.006 (0.940)

17 40 53 17 48 33 0.004 (0.949)

sin

thismeta-analysis

vs.AA

GAvs.AA

GG/GAvs.AA

GG

vs.GA/AA

Ph

I2(%

)OR(95%

CI)

Ph

I2(%

)OR(95%

CI)

Ph

I2(%

)OR

(95%

CI)

Ph

I2(%

)

32)

0.00

71.6

1.153(0.858e1.550)

0.000

68.7

1.181(0.873e1.599)

0.000

70.9

1.148

(0.903e

1.459)

0.004

61.1

46)

0.000

80.5

1.151(0.716e1.849)

0.000

83.4

1.114(0.689e1.803)

0.000

85.6

0.970

(0.760e

1.240)

0.101

45.8

93)

0.405

0.0

1.268(0.860e1.871)

0.424

0.0

1.202(0.833e1.733)

0.368

0.0

0.908

(0.560e

1.472)

0.558

0.00

21)

0.492

0.00

1.135(0.735e1.753)

0.647

0.00

1.418(0.941e2.136)

0.667

0.00

1.758

(1.271e

2.433

)0.294

18.3

32)

0.036

61.0

1.432(1.078e1.902)

0.173

37.2

1.370(0.975e1.926)

0.040

60.1

1.088

(0.745e

1.589)

0.187

35.1

05)

0.001

75.9

0.970(0.626e1.503)

0.007

68.7

1.056(0.675e1.653)

0.002

73.5

1.199

(0.866e

1.660)

0.002

74.2

itytest;PB,population-based

study;HBhospital-based

study.

237CTLA-4 Exon-1 +49AOG Polymorphism and Primary Biliary Cirrhosis Risk

separately according to the different subgroups. Therefore,each group in one publication was considered separatelyfor subgroup analysis. Hence, a total of 11 studies including2151 cases and 2214 controls were involved in this meta-analysis. Table 1 shows the detailed characteristics of thesestudies. Of the 11 studies, sample sizes ranged from 117 to863. Among these studies, four were hospital-based (1405cases and 1355 controls) and seven were population-based(746 cases and 859 controls). There were two studies ofmixed populations (230 cases and 269 controls), six studiesfrom Caucasians (1536 cases and 1517 controls), and threestudies of Asians (385 cases and 428 controls).

Meta-analysis

The results of the association between CTLA þ49GOApolymorphism and PBC, along with the heterogeneity testare shown in Table 2. Overall, no significant associationsbetweenCTLAþ49GOApolymorphism and PBC riskwerefound for GAvs. AA (OR5 1.190; 95% CI5 0.818e1.732;p5 0.000 for heterogeneity), GG vs. AA (OR5 1.153; 95%CI5 0.858e1.550; p5 0.000 for heterogeneity), GGþGAvs. AA (OR5 1.181; 95% CI5 0.873e1.599; p5 0.000 forheterogeneity) and GG vs. GA þ AA (OR 5 1.148; 95%CI5 0.903e1.459; p5 0.004 for heterogeneity). However,in the subgroup analysis by ethnicity, significant associationswere observed in Asians for GG vs. AA (OR 5 1.873; 95%CI 5 1.202e2.921; p 5 0.492 for heterogeneity) and GGvs. AA þ GA (OR 5 1.758; 95% CI 5 1.271e2.433; p 50.294 for heterogeneity). Moreover, in the stratified analysisby control sources, a significant association was observed inpopulation-based studies for GA vs. AA (OR 5 1.432; 95%CI 5 1.078e1.902; p 5 0.173 for heterogeneity).

Publication Bias and Sensitivity Analysis

Begg’s funnel plot and Egger’s test were performed toaccess the publication bias of literatures. The shape of thefunnel plot did not reveal obvious asymmetry (figures notshown) and the Egger’s test suggested the absence of publi-cation bias ( p 5 0.372 for GA vs. AA; p 5 0.787 for GGvs. AA; p 5 0.971 for dominant model; p 5 0.608 forrecessive model). Sensitivity analysis was carried out byincluding and excluding one study not in HWE. The pooledORs were not materially altered (data not shown), implyingthat our results were robust.

Table

2.MainresultsofpooledOR

Studygroups

GG

OR

(95%

CI)

Total

1.190

(0.818e

1.7

Ethnicity

Caucasian

1.018

(0.593e

1.7

Mixed

1.052

(0.618e

1.7

Asian

1.873

(1.202e

2.9

Source

PB

1.190

(0.818e

1.7

HB

1.168

(0.756e

1.8

Ph,pvalueofQ-testforheterogene

Discussion

In this studywe performed a systematic review of associationbetween the CTLA-4þ49GOA polymorphism and risk forPBC based on eight caseecontrol studies for which informa-tion was available. With 2151 patients with PBC and 2214controls included, this study had greater power than allprevious studies. Significant associations were detected inAsians but not in the global analysis. This result suggested

238 Huang et al./ Archives of Medical Research 42 (2011) 235e238

the differences in genetic backgrounds and the environmentthey lived in may influence the association between theCTLA-4 þ49GOA polymorphism and PBC risk.

Our results indicated that GG vs. GA/AA genotypesincreased PBC risk among population-based studies butnot among hospital-based studies. This may be becausehospital-based studies have a high risk of producing unreli-able results because hospital-based controls may not alwaysbe truly representative of the general population. Therefore,a methodologically preferable design such as using a properand representative population-based study is crucial toavoid selection bias.

The current meta-analysis has some strength comparedwith previous meta-analyses (27). First, different fromother meta-analyses, we performed a meta-analysis in threedifferent genetic models. Second, subgroup analyses wereperformed by ethnicities and sources of controls to obtaina more precise estimation of the association. Third, sensi-tivity analyses were performed pooling with and withoutstudies not in HWE to test the robustness of the results.

However, some limitations of this meta-analysis shouldbe acknowledged. First, the overall outcomes were basedon unadjusted estimates, whereas a more precise evaluationshould be adjusted by other covariants including age, bodymass index, gender, ethnicity, alcohol habit, and environ-ment factors. It will also be helpful to understand thedifferent distributions of other known or unknown riskfactors that may interact with genetic factors in develop-ment of PBC. Second, some controls were selected fromhospital-based populations and not uniformly defined.Control groups may have different risks of developingPBC. Third, phenotype characters would greatly influencestudy results. Some studies have shown that PBC patientsoften coexist with other autoimmune diseases, which maybe risk factors in development of PBC. Last but not least,the number of Asians and Africans was relatively small.This makes results from the subgroup analysis less reliable.

In conclusion, this meta-analysis suggests that theCTLA-4 þ49GOA polymorphism may be a risk factorfor PBC in Asians. More studies or large case-controlstudies should be performed to clarify its possible role inAsian and African populations.

References1. Kaplan MM, Gershwin ME. Primary biliary cirrhosis. N Engl J Med

2005;353:1261e1273.

2. Jones DE. Pathogenesis of primary biliary cirrhosis. J Hepatol 2003;

39:639e648.

3. Gershwin ME, Selmi C, Worman HJ, et al. Risk factors and comorbid-

ities in primary biliary cirrhosis: a controlled interview-based study of

1032 patients. Hepatology 2005;42:1194e1202.

4. Gershwin ME, Mackay IR. The causes of primary biliary cirrhosis:

convenient and inconvenient truths. Hepatology 2008;47:737e745.5. Underhill J, Donaldson P, Bray G, et al. Susceptibility to primary

biliary cirrhosis is associated with the HLA-DR8-DQB1*0402 haplo-

type. Hepatology 1992;16:1404e1408.

6. Gregory WL, Mehal W, Dunn AN, et al. Primary biliary cirrhosis:

contribution of HLA class II allele DR8. Q J Med 1993;86:393e399.

7. Manns MP, Bremm A, Schneider PM, et al. HLA DRw8 and comple-

ment C4 deficiency as risk factors in primary biliary cirrhosis. Gastro-

enterology 1991;101:1367e1373.

8. Mehal WZ, Gregory WL, Lo YM, et al. Defining the immunogenetic

susceptibility to primary biliary cirrhosis. Hepatology 1994;20:

1213e1219.

9. Maeda T, Onishi S, Saibara T, et al. HLA DRw8 and primary biliary

cirrhosis. Gastroenterology 1992;103:1118e1119.

10. Onishi S, Sakamaki T, Maeda T, et al. DNA typing of HLA class II

genes: DRB1*0803 increases the susceptibility of Japanese to primary

biliary cirrhosis. J Hepatol 1994;21:1053e1060.11. Hirschfield GM, Liu X, Xu C, et al. Primary biliary cirrhosis associ-

ated with HLA, IL12A, and IL12RB2 variants. N Engl J Med 2009;

360:2544e2555.

12. Joshita S, Umemura T, Yoshizawa K, et al. A2BP1 as a novel suscep-

tible gene for primary biliary cirrhosis in Japanese patients. Hum

Immunol 2010;71:520e524.

13. Dariavach P, Mattei MG, Golstein P, et al. Human Ig superfamily

CTLA-4 gene: chromosomal localization and identity of protein

sequence between murine and human CTLA-4 cytoplasmic domains.

Eur J Immunol 1988;18:1901e1905.

14. Nistico L, Buzzetti R, Pritchard LE, et al. The CTLA-4 gene region of

chromosome 2q33 is linked to, and associated with, type 1 diabetes.

Belgian Diabetes Registry. Hum Mol Genet 1996;57:1075e1080.

15. Donaldson P, Veeramani S, BaragiottaA, et al. Cytotoxic T-lymphocyte-

associated antigen-4 single nucleotide polymorphisms andhaplotypes in

primary biliary cirrhosis. Clin Gastroenterol Hepatol 2007;5:755e760.

16. Bittencourt PL, Pal�acios SA, Farias AQ, et al. Analysis of major

histocompatibility complex and CTLA-4 alleles in Brazilian patients

with primary biliary cirrhosis. J Gastroenterol Hepatol 2003;18:

1061e1066.

17. Schott E, Witt H, Pascua M, et al. Association of CTLA4 single nucle-

otide polymorphisms with viral but not autoimmune liver disease. Eur

J Gastroenterol Hepatol 2007;19:947e951.

18. Fan LY, Tu XQ, Cheng QB, et al. Cytotoxic T lymphocyte associated

antigen-4 gene polymorphisms confer susceptibility to primary biliary

cirrhosis and autoimmune hepatitis in Chinese population. World J

Gastroenterol 2004;10:3056e3059.

19. Oertelt S, Kenny TP, Selmi C, et al. SNP analysis of genes implicated

in T cell proliferation in primary biliary cirrhosis. Clin Dev Immunol

2005;12:259e263.20. Joshita S, Umemura1 T, Yoshizawa K, et al. Association analysis of

cytotoxic T-lymphocyte antigen 4 gene polymorphisms with primary

biliary cirrhosis in Japanese patients. J Hepatol 2010;53:537e541.21. Agarwal K, Jones DEJ, Daly AK, et al. CT.A-4 gene polymorphism

confers susceptibility to primary biliary cirrhosis. J Hepatol 2000;32:

538e541.

22. Juran BD, Atkinson EJ, Larson JJ, et al. Carriage of a tumor necrosis

factor polymorphism amplifies the cytotoxic T-lymphocyte antigen 4

attributed risk of primary biliary cirrhosis: evidence for a geneegene

interaction. Hepatology 2010;52:223e229.

23. Cochran WG. The combination of estimates from different experi-

ments. Biometrics 1954;10:101e129.

24. Mantel N, Haenszel W. Statistical aspects of the analysis of data from

retrospective studies of disease. J Natl Cancer Inst 1959;22:719e748.

25. DerSimonian R, Laird N. Meta-analysis in clinical trials. Control Clin

Trials 1986;7:177e188.

26. ThakkinstianA,McElduff P,D’EsteC, et al. Amethod formeta-analysis

of molecular association studies. Stat Med 2005;24:1291e1306.27. Miyake Y, Ikeda F, Takaki A, et al. þ49A/G polymorphism of cyto-

toxic T-lymphocyte antigen 4 gene in type 1 autoimmune hepatitis

and primary biliary cirrhosis: a meta-analysis. Hepatol Res 2011;41:

151e159.