- Email: [email protected]
Investigation of cytotoxic T-lymphocyte-associated protein 4 gene polymorphisms in symptomatic gallstone disease
Human Immunology 72 (2011) 355–358
Contents lists available at ScienceDirect
Investigation of cytotoxic T-lymphocyte-associated protein 4 gene polymorphisms in symptomatic gallstone disease Shou-Chuan Shih a,b, Horng-Woei Yang c, Tzu-Yang Chang c, Kuang-Chun Hu a,b, Shih-Chuan Chang c, Chiung-Ling Lin c, Chien-Yuan Hung a, Horng-Yuan Wang a, Marie Lin c, Yann-Jinn Lee c,d,e,* a
Department of Internal Medicine, Mackay Memorial Hospital, Taipei, Taiwan Mackay Medicine, Nursing and Management College, Taipei, Taiwan c Department of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan d Department of Pediatrics, Mackay Memorial Hospital, Taipei, Taiwan e Department of Pediatrics, Taipei Medical University, Taipei, Taiwan b
A R T I C L E
I N F O
Article history: Received 19 May 2010 Accepted 13 January 2011 Available online 25 January 2011
Keywords: CTLA4 Gallstone disease Taiwanese Single nucleotide polymorphism Case– control study
A B S T R A C T
Gallstone disease (GSD), which is increasingly prevalent in Taiwan, develops through a complex process involving genetic, environmental, and immune factors. Cytotoxic T-lymphocyte-associated protein 4 (CTLA4) limits T-cell proliferation. The present study looked for associations between symptomatic GSD and polymorphisms of the CTLA4 gene. For this case– control cross-sectional study among Taiwanese, 275 patients with symptomatic GSD and 852 controls were enrolled. Genotyping of CTLA4⫺318 C/T, +49 A/G, and CT60 A/G single nucleotide polymorphisms (SNPs) was performed by polymerase chain reaction–restriction fragment length polymorphism. The genotype, allele, carrier, and haplotype frequencies were calculated by direct counting or with Haploview 4.1 software. Genotype, allele, carrier, and haplotype frequencies of the CTLA4 SNPs studied were equally distributed in symptomatic GSD patients and controls. No significant associations between symptomatic GSD and these 3 SNPs were observed. Our data suggest that CTLA4⫺318 C/T, +49 A/G, and CT60 A/G SNPs do not confer increased susceptibility to symptomatic GSD. 䉷 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction Gallstone disease (GSD) is common in most European countries as well as in North and South America, with a prevalence of more than 10% [1– 4]. Epidemiologic surveys in Taiwan indicate a dramatic increase in the prevalence of GSD over the past 16 years from 4.3 to 10.7%, with patients presenting with acute cholecystitis, acute cholangitis, or biliary pancreatitis [5–7]. The overall prevalence of asymptomatic GSD was 5% and no significant difference in gender in a community-based study was found in Taiwan [6]. These data indicate that GSD has become the most serious gastrointestinal problem in Taiwan [8]. Gallstone biogenesis results from complicated interactions between genetic factors and high-carbohydrate, high-cholesterol, or low-fiber diets [9]. The documented risk factors of GSD include older age, female gender, high-calorie low-fiber diet, obesity, and low physical activity [10]. Recently, immune system and immunerelated genes were implicated in the gallstone formation [11–14]. Immunoglobulins, especially IgM, IgA, and IgG, not only are involved in inflammatory disorders but also contribute to the nucleation of supersaturated cholesterol in the bile and crystal formation
* Corresponding author. E-mail address: [email protected] (Dr Y.-J. Lee).
[12,13]. A murine study has also demonstrated a crucial role of T cells in GSD [14]. Another study of interleukin 4 (IL-4)-deficient mice exhibited enhanced gallstone formation in mice fed a highcholesterol diet [11]. Inflammation resulting from a series of immune responses may lead to gallstone-related symptoms [10]. In addition, inflammatory responses were involved in different types of gallstones [15,16]. However, an association between GSD and genes in the immune response pathways has not yet been conclusively demonstrated [17,18]. Given the complexity of the immune response, investigation of other immune-related genes is needed to understand genetic susceptibility to GSD. The cytotoxic T-lymphocyte-associated protein 4 gene (CTLA4) is located on chromosome 2q33 and contains 4 exons. CTLA4 is a member of the immunoglobulin superfamily that inhibits T-cell proliferation [19]. In view of the critical role of CTLA4 in regulation of the immune system [20] and the contribution of immune and inflammatory reactions to the formation of different types of gallstones [14], we hypothesized that there might be a genetic association between CTLA4 single nucleotide polymorphisms (SNPs) and GSD. We therefore analyzed 3 of the most common SNPs in the CTLA4 gene, ⫺318 C/T (rs5742909), +49 A/G (rs231775), and CT60 A/G (rs3087243), in Taiwanese patients with symptomatic GSD compared with healthy control subjects.
0198-8859/11/$32.00 - see front matter 䉷 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2011.01.004
356
S.-C. Shih et al. / Human Immunology 72 (2011) 355–358
Table 1 Genotype, allele, and carrier frequencies of CTLA4⫺318 C/T polymorphism in patients with GSD and controls Frequencies
Genotypea C/C C/T T/T Alleleb C T Carrierc C T
GSD n ⫽ 275 (%)
Control n ⫽ 852 (%)
OR (95% CI)
2
p
pc
234 (85.1) 36 (13.1) 5 (1.8)
691 (81.1) 152 (17.8) 9 (1.1)
1.33 (0.92–1.93) 0.69 (0.47–1.03) 1.73 (0.58–5.22)
2.25 3.37 0.98
0.13 0.06 0.32
0.25 0.13 0.54
504 (91.6) 46 (8.4)
1534 (90.0) 170 (10.0)
1.21 (0.86–1.71) 0.82 (0.59–1.16)
1.25 1.25
0.26 0.26
0.26 0.26
270 (98.2) 41 (14.9)
843 (98.9) 161 (18.9)
0.58 (0.19–1.74) 0.75 (0.52–1.09)
0.98 2.25
0.32 0.13
0.32 0.13
⫽ 4.19, p ⫽ 0.12 (3 ⫻ 2 contingency table). ⫽ 1.25, p ⫽ 0.26 (2 ⫻ 2 contingency table). ⫽ 1.49, p ⫽ 0.22 (2 ⫻ 2 contingency table). Data are in n (%). OR ⫽ odds ratio, 95% CI ⫽ 95% confidence interval. Hardy–Weinberg test for patients: p ⫽ 0.058; for controls: p ⫽ 0.95.
a 2
b 2 c 2
Table 2 Genotype, allele, and carrier frequencies of CTLA4 +49 A/G polymorphism in patients with GSD and controls Frequencies
Genotypea A/A A/G G/G Alleleb A G Carrierc A G
GSD n ⫽ 275 (%)
Control n ⫽ 852 (%)
OR (95% CI)
2
p
pc
25 (9.1) 122 (44.4) 128 (46.5)
89 (10.4) 381 (44.7) 382 (44.8)
0.86 (0.54–1.37) 0.99 (0.75–1.30) 1.07 (0.82–1.41)
0.42 0.01 0.25
0.52 0.92 0.62
0.77 0.99 0.86
172 (31.3) 378 (68.7)
559 (32.8) 1145 (67.2)
0.93 (0.76–1.15) 1.07 (0.87–1.32)
0.45 0.45
0.50 0.50
0.50 0.50
147 (53.5) 250 (90.9)
470 (55.2) 763 (89.6)
0.93 (0.71–1.23) 1.17 (0.73–1.86)
0.25 0.42
0.62 0.52
0.62 0.52
⫽ 0.52, p ⫽ 0.77 (3 ⫻ 2 contingency table). ⫽ 0.45, p ⫽ 0.50 (2 ⫻ 2 contingency table). c 2 ⫽ 0.15, p ⫽ 0.70 (2 ⫻ 2 contingency table). Data are in n (%). OR ⫽ odds ratio, 95% CI ⫽ 95% confidence interval. Hardy–Weinberg test for patients: p ⫽ 0.72; for controls: p ⫽ 0.75. a 2
b 2
2. Patients and methods 2.1. Patients and control subjects We enrolled 275 unrelated Taiwanese patients (125 men, 150 women) with mixed and symptomatic gallstones. GSD was diagnosed on the basis of clinical, laboratory, and imaging evidence or surgical proof. The average age at diagnosis was 52.7 ⫾ 13.0 years (23.5– 83.9 years). The control group included 852 Taiwanese (411 men, 441 women), either hospital personnel or individuals undergoing routine health examinations or minor surgery. None had a history of biliary disorders but may have had asymptomatic gallstones. Our institutional review board approved this study, and all subjects gave informed consent.
Beverly, MA), respectively. The ⫺318 C/T SNP was amplified with modified primers 5=-GTTAGGGATGCCCAGAAGAT- 3= and 5=-CTCAACTGAACAAAACAAGC-3=, resulting in a 172-bp product [23]. The +49 A/G SNP was amplified with primers 5=-AAGGCTCAGCTGAACCTGGT-3= and 5=-CTGCTGAAACAAATGAAACCC-3=, resulting in a 153-bp product [24]. The CT60 A/G SNP was amplified with primers 5=-CACCACTATTTGGGATATACC-3= and 5=-AGGTCTATATTTCAGGAAGGC-3=, resulting in a 216-bp product [25]. The individual amplified products were digested with restriction enzymes and separated on a 3.5% agarose gel (SeaKem LE agarose, Cambrex, Rockland, ME) as described in our previous study [26]. 2.5. Statistical power
dbSNP rs5742909 (⫺318 C/T) is a transition at position ⫺318 of the promoter region, rs231775 (+49 A/G) is in exon 1 at position ⫹49, and rs3087243 (CT60 A/G) at position ⫹6230 [21]. The CT60 G allele is associated with a 50% decrease in the soluble CTLA4 isoform [21]. Individuals with the ⫺318 T allele or ⫹49 G/G genotype have higher CTLA4 expression than those without these genotypes [22].
Because the asymptomatic cholelithic cases hidden in controls may influence the authenticity of our study, we adopted the feature of unselected controls built into Genetic Power Calculator software [27]. With this feature, which indicates a true random population sample (e.g., for a 1% disease, 1% of controls would also, by chance, have the disease), we designed the study to have ⬎80% power in a case– control study at a 5% significance level to detect a relative risk of 1.9 for GSD with a particular genotype and an estimated prevalence of 0.05 [6,28].
2.3. DNA extraction
2.6. Statistical analysis
DNA was extracted from fresh or frozen peripheral blood leukocytes from GSD patients and controls using standard methods.
The genotype, allele, and carrier frequencies of the ⫺318 C/T, +49 A/G, and CT60 A/G SNPs were determined by direct counting. Hardy–Weinberg equilibrium was assessed for each SNP in both patient and control groups with Haploview 4.1 software [29]. The genotype, allele, and carrier distributions of patients and controls were compared by a 2 test (3 ⫻ 2 or 2 ⫻ 2 contingency tables) with Yates’ correction where appropriate (one expected number ⬍ 5).
2.2. SNP selection
2.4. CTLA4 genotyping ⫺318 C/T, +49 A/G, and CT60 A/G SNPs were typed by polymerase chain reaction–restriction fragment length polymorphism using MseI, BstEII, and NcoI restriction enzymes (New England BioLabs,
S.-C. Shih et al. / Human Immunology 72 (2011) 355–358
357
Table 3 Genotype, allele, and carrier frequencies of CTLA4 CT60 A/G polymorphism in patients with GSD and controls Frequencies
Genotypea A/A A/G G/G Alleleb A G Carrierc A G
OR (95% CI)
2
p
pc
36 (4.2) 302 (35.5) 514 (60.3)
1.12 (0.59–2.15) 0.92 (0.69–1.22) 1.06 (0.81–1.41)
0.13 0.36 0.19
0.72 0.55 0.66
0.92 0.79 0.88
118 (21.5) 432 (78.5)
374 (21.9) 1330 (78.1)
0.97 (0.77–1.23) 1.03 (0.81–1.30)
0.06 0.06
0.81 0.81
0.81 0.81
105 (38.2) 262 (95.3)
338 (39.7) 816 (95.8)
0.94 (0.71–1.24) 0.89 (0.46–1.70)
0.19 0.13
0.66 0.72
0.66 0.72
GSD n ⫽ 275 (%)
Control n ⫽ 852 (%)
13 (4.7) 92 (33.5) 170 (61.8)
⫽ 0.43, p ⫽ 0.81 (3 ⫻ 2 contingency table). ⫽ 0.59E-01, p ⫽ 0.81 (2 ⫻ 2 contingency table). ⫽ 0.62E-01, p ⫽ 0.80 (2 ⫻ 2 contingency table). Data are in n (%). OR ⫽ odds ratio, 95% CI ⫽ 95% confidence interval. Hardy–Weinberg test for patients: p ⫽ 1.0; for controls: p ⫽ 0.37.
a 2
b 2 c 2
Table 4 Analysis of CTLA4 haplotypes in patients with GSD and controls Haplotypea
Patients 2N ⫽ 550 (%)
Controls 2N ⫽ 1074 (%)
OR (95% CI)
2
p
pc
CGG CAA TAG CAG
377.85 (68.7) 118.25 (21.5) 46.2 (8.4) 8.25 (1.5)
707.77 (65.9) 222.32 (20.7) 100.96 (9.4) 25.78 (2.4)
1.14 (0.91–1.42) 1.05 (0.82–1.35) 0.88 (0.61–1.27) 0.62 (0.28–1.37)
1.29 0.14 0.44 1.44
0.26 0.71 0.51 0.23
0.45 0.91 0.76 0.41
a p value for 4 haplotypes between patients and controls: p ⫽ 0.48 (2 ⫽ 2.49, 3 df) (4 ⫻ 2 contingency table). Haplotype inferred using Haploview 4.1, based on the ⫺318 C/T, +49 A/G, and CT60 A/G SNPs. OR ⫽ odds ratio, 95% CI ⫽ 95% confidence interval.
Odds ratios (OR) and 95% confidence intervals (95% CI) for particular genotypes, alleles, and carriers associated with the risk of GSD were calculated [30]. Frequencies of CTLA4 haplotypes (⫺318 C/T, +49 A/G, and CT60 A/G) in patients and in controls were estimated using Haploview 4.1. Linkage disequilibrium between SNPs among controls was analyzed with Haploview 4.1. Statistical differences, OR, and 95% CI for the association of various haplotypes with GSD were determined. The Bonferroni correction was used to correct for multiple comparisons where appropriate. pc values of less than 0.05 (two-tailed) were considered statistically significant. 3. Results The genotype distributions of the ⫺318 C/T, +49 A/G, and CT60 A/G SNPs in GSD patients and controls were in Hardy–Weinberg equilibrium (p ⬎ 0.4). The frequencies of genotypes, alleles, and carriers did not differ significantly between patients and controls (Tables 1–3) or by gender (data not shown). Among controls, there was strong linkage disequilibrium between ⫺318 C/T and +49 A/G (D= ⫽ 0.954, r2 ⫽ 0.207), +49 A/G and CT60 A/G (D= ⫽ 0.932, r2 ⫽ 0.5), and ⫺318 C/T and CT60 A/G (D= ⫽ 0.856, r2 ⫽ 0.023; Suppl. Fig. 1). Haplotype CGG was the most prevalent among the 4 observed. The haplotype frequencies did not differ between patients and controls (Table 4) or between men and women (data not shown). 4. Discussion Our study showed no association between the SNPs of the CTLA4 gene investigated and GSD in Taiwanese patients. There were no differences in genotype, allele, or carrier frequencies between patients with GSD and controls. A haplotype consists of a specific combination of alleles along a chromosome [31]. It contains several polymorphic sites to produce a phenotype [32–34] and is more clearly linked with a functional polymorphism than individual markers [35,36]. Therefore, haplotype-based analysis may be more powerful in detecting certain associations [36]. However, we also failed to detect a significant difference in haplotype frequencies
between patients and controls, further confirming the lack of association among these 3 CTLA4 SNPs and GSD. Mechanisms contributing to the formation of gallstones include the cholesterol and bilirubin concentrations in bile, impaired gallbladder motility, and destabilization of bile by kinetic protein factors [8,37], each of which may promote gallstone formation. Bile salt crystals and ablated gallbladder epithelial cells form proinflammatory elements that promote induction of T-cell-dependent cytokines and lead to an inflammatory response [38]. The inflammation may further interfere with gallbladder motility and alter bile emptying [38]. In the previous study, transplantation of T cells into T-cell-deficient mice significantly increased cholesterol crystals and mucin gel in the gallbladder when the mice were fed a lithogenic diet. The treated mice also had an increase in Th1-related cytokines, such as interleukin 1, interferon-␥, and tumor necrosis factor-␣ [14]. Tumor necrosis factor-␣ may induce the overproduction of MUC5AC protein and enhance mucin secretion in human gallbladder [39]. Mucin gel can agglomerate with bile salt or cholesterol crystals into gallstones. These data demonstrate that T-cell activation and Th1-related immunity are important in gallstone biogenesis. Although the pathogeneses of cholesterol and pigment gallstones are different [40,41], inflammation responses may be involved in gallstone formation of both types [15,42,43], which is why we hypothesized that genes involved in immune responses might be associated with GSD. The CTLA4 protein contains a leader sequence, a V-domain, a transmembrane domain, and a cytoplasmic tail. In conjunction with CD28, the CTLA4 protein regulates T-cell responses to maintain proper T-cell balance [23]. CTLA4 is expressed on activated T cells and then binds to B7-1 (CD80) and B7-2 (CD86) on antigenpresenting cells and transmits inhibitory signals to repress overactivation of T cells [44,45]. Ctla4-/-mice exhibit T-cell overproliferation and early death [46]. SNP -318 C/T in the promoter region alters transcription of the CTLA4 gene [47], whereas SNP ⫹49 A/G affects posttranslational modification [48], and SNP CT60 A/G limits efficient production of soluble CTLA4 mRNA [21]. If these polymor-
358
S.-C. Shih et al. / Human Immunology 72 (2011) 355–358
phisms were combined, the synergistic effect resulting in increased T-cell activity might well contribute to a variety of diseases [21]. We found no association between GSD and the 3 SNPs studied. That does not rule out the possibility that other downstream sites in the CTLA4 signaling pathway are involved in GSD susceptibility. CTLA4 regulates transcription factor FOXP3⫹ activity in the CD4⫹ pathway [46] and triggers eomesodermin, a transcription factor that activates interferon-␥ and granzyme B activity in a CD8⫹dependent manner [49]. Such signaling sites are candidates for investigation for a possible role in altering the risk of GSD. Our study may still have the possibility of a type II (false-negative) error, although this is unlikely because the study was designed to have over 80% power to detect a significant association between the SNPs studied [27]. Therefore, extensive genotyping with more SNPs, especially with haplotype tag SNPs in the CTLA4 gene, is warranted. Acknowledgments We thank Dr Mary Jeanne Buttrey for her review and criticism of our manuscript. This study was supported by Grants MMH 9362 and E-9807 from Mackay Memorial Hospital, Taipei, Taiwan. Appendix. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.humimm.2011.01.004. References [1] Borch K, Jonsson KA, Zdolsek JM, Halldestam I, Kullman E. Prevalence of gallstone disease in a Swedish population sample. Relations to occupation, childbirth, health status, life style, medications, and blood lipids. Scand J Gastroenterol 1998;33:1219 –25. [2] Everhart JE, Khare M, Hill M, Maurer KR. Prevalence and ethnic differences in gallbladder disease in the United States. Gastroenterology 1999;117:632–9. [3] Everhart JE, Yeh F, Lee ET, Hill MC, Fabsitz R, Howard BV, et al. Prevalence of gallbladder disease in American Indian populations: findings from the Strong Heart Study. Hepatology 2002;35:1507–12. [4] Shaffer EA. Gallstone disease: epidemiology of gallbladder stone disease. Best Pract Res Clin Gastroenterol 2006;20:981–96. [5] Lu SN, Chang WY, Wang LY, Hsieh MY, Chuang WL, Chen SC, et al. Risk factors for gallstones among Chinese in Taiwan. A community sonographic survey. J Clin Gastroenterol 1990;12:542– 6. [6] Chen CH, Huang MH, Yang JC, Nien CK, Etheredge GD, Yang CC, et al. Prevalence and risk factors of gallstone disease in an adult population of Taiwan: an epidemiological survey. J Gastroenterol Hepatol 2006;21:1737– 43. [7] Huang J, Chang CH, Wang JL, Kuo HK, Lin JW, Shau WY, et al. Nationwide epidemiological study of severe gallstone disease in Taiwan. BMC Gastroenterol 2009;9:63. [8] Ho KJ, Lin XZ, Yu SC, Chen JS, Wu CZ. Cholelithiasis in Taiwan. Gallstone characteristics, surgical incidence, bile lipid composition, and role of betaglucuronidase. Dig Dis Sci 1995;40:1963–73. [9] Lammert F, Wang DQ. New insights into the genetic regulation of intestinal cholesterol absorption. Gastroenterology 2005;129:718 –34. [10] Lammert F, Miquel JF. Gallstone disease: from genes to evidence-based therapy. J Hepatol 2008;48 (suppl 1):S124 –35. [11] King VL, Szilvassy SJ, Daugherty A. Interleukin-4 deficiency promotes gallstone formation. J Lipid Res 2002;43:768 –71. [12] Harvey PR, Upadhya GA, Strasberg SM. Immunoglobulins as nucleating proteins in the gallbladder bile of patients with cholesterol gallstones. J Biol Chem 1991;266:13996 – 4003. [13] Upadhya GA, Harvey PR, Strasberg SM. Effect of human biliary immunoglobulins on the nucleation of cholesterol. J Biol Chem 1993;268:5193–200. [14] Maurer KJ, Rao VP, Ge Z, Rogers AB, Oura TJ, Carey MC, et al. T-cell function is critical for murine cholesterol gallstone formation. Gastroenterology 2007; 133:1304 –15. [15] Rege RV, Prystowsky JB. Inflammatory properties of bile from dogs with pigment gallstones. Am J Surg 1996;171:197–201. [16] Rege RV, Prystowsky JB. Inflammation and a thickened mucus layer in mice with cholesterol gallstones. J Surg Res 1998;74:81–5. [17] Shih SC, Lee YJ, Liu HF, Dang CW, Chang SC, Lin SC, et al. Polymorphism in transmembrane region of MICA gene and cholelithiasis. World J Gastroenterol 2003;9:1541– 4. [18] Papasteriades C, al-Mahmoud I, Papageorgakis N, Romania S, Katsas A, Ollier W, et al. HLA antigens in Greek patients with cholelithiasis. Dis Markers 1990;8:17–21. [19] Alegre ML, Frauwirth KA, Thompson CB. T-cell regulation by CD28 and CTLA-4. Nat Rev Immunol 2001;1:220 – 8.
[20] McCoy KD, Le Gros G. The role of CTLA-4 in the regulation of T cell immune responses. Immunol Cell Biol 1999;77:1–10. [21] Ueda H, Howson JM, Esposito L, Heward J, Snook H, Chamberlain G, et al. Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease. Nature 2003;423:506 –11. [22] Ligers A, Teleshova N, Masterman T, Huang WX, Hillert J. CTLA-4 gene expression is influenced by promoter and exon 1 polymorphisms. Genes Immun 2001;2:145–52. [23] Harper K, Balzano C, Rouvier E, Mattei MG, Luciani MF, Golstein P. CTLA-4 and CD28 activated lymphocyte molecules are closely related in both mouse and human as to sequence, message expression, gene structure, and chromosomal location. J Immunol 1991;147:1037– 44. [24] Marron MP, Raffel LJ, Garchon HJ, Jacob CO, Serrano-Rios M, Martinez Larrad MT, et al. Insulin-dependent diabetes mellitus (IDDM) is associated with CTLA4 polymorphisms in multiple ethnic groups. Hum Mol Genet 1997;6: 1275– 82. [25] Torres B, Aguilar F, Franco E, Sanchez E, Sanchez-Roman J, Jimenez Alonso J, et al. Association of the CT60 marker of the CTLA4 gene with systemic lupus erythematosus. Arthritis Rheum 2004;50:2211–5. [26] Su TH, Chang TY, Lee YJ, Chen CK, Liu HF, Chu CC, et al. CTLA-4 gene and susceptibility to human papillomavirus-16-associated cervical squamous cell carcinoma in Taiwanese women. Carcinogenesis 2007;28:1237– 40. [27] Purcell S, Cherny SS, Sham PC. Genetic Power Calculator: design of linkage and association genetic mapping studies of complex traits. Bioinformatics 2003; 19:149 –50. [28] Buch S, Schafmayer C, Volzke H, Becker C, Franke A, von Eller-Eberstein H, et al. A genome-wide association scan identifies the hepatic cholesterol transporter ABCG8 as a susceptibility factor for human gallstone disease. Nat Genet 2007; 39:995–9. [29] Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 2005;21:263–5. [30] Lee YJ, Chen MR, Chang WC, Lo FS, Huang FY. A freely available statistical program for testing associations. MD Comput 1998;15:327–30. [31] International HapMap Consortium. A haplotype map of the human genome. Nature 2005;437:1299 –320. [32] Drysdale CM, McGraw DW, Stack CB, Stephens JC, Judson RS, Nandabalan K, et al. Complex promoter and coding region beta 2-adrenergic receptor haplotypes alter receptor expression and predict in vivo responsiveness. Proc Natl Acad Sci U S A 2000;97:10483– 8. [33] Joosten PH, Toepoel M, Mariman EC, Van Zoelen EJ. Promoter haplotype combinations of the platelet-derived growth factor alpha-receptor gene predispose to human neural tube defects. Nat Genet 2001;27:215–7. [34] Cowles CR, Hirschhorn JN, Altshuler D, Lander ES. Detection of regulatory variation in mouse genes. Nat Genet 2002;32:432–7. [35] Kruglyak L. The use of a genetic map of biallelic markers in linkage studies. Nat Genet 1997;17:21– 4. [36] Zaykin DV, Westfall PH, Young SS, Karnoub MA, Wagner MJ, Ehm MG. Testing association of statistically inferred haplotypes with discrete and continuous traits in samples of unrelated individuals. Hum Hered 2002;53:79 –91. [37] Paumgartner G, Sauerbruch T. Gallstones: pathogenesis. Lancet 1991;338: 1117–21. [38] van Erpecum KJ, Wang DQ, Moschetta A, Ferri D, Svelto M, Portincasa P, et al. Gallbladder histopathology during murine gallstone formation: relation to motility and concentrating function. J Lipid Res 2006;47:32– 41. [39] Finzi L, Barbu V, Burgel PR, Mergey M, Kirkwood KS, Wick EC, et al. MUC5AC, a gel-forming mucin accumulating in gallstone disease, is overproduced via an epidermal growth factor receptor pathway in the human gallbladder. Am J Pathol 2006;169:2031– 41. [40] Cahalane MJ, Neubrand MW, Carey MC. Physical– chemical pathogenesis of pigment gallstones. Semin Liver Dis 1988;8:317–28. [41] Carey MC. Pathogenesis of gallstones. Am J Surg 1993;165:410 –9. [42] Prystowsky JB, Huprikar JS, Rademaker AW, Rege RV. Human polymorphonuclear leukocyte phagocytosis of crystalline cholesterol, bilirubin, and calcium hydroxyapatite in vitro. Dig Dis Sci 1995;40:412– 8. [43] Prystowsky JB, Rege RV. The inflammatory effects of crystalline cholesterol monohydrate in the guinea pig gallbladder in vivo. Surgery 1998;123:258 – 63. [44] Manzotti CN, Tipping H, Perry LC, Mead KI, Blair PJ, Zheng Y, et al. Inhibition of human T cell proliferation by CTLA-4 utilizes CD80 and requires CD25⫹ regulatory T cells. Eur J Immunol 2002;32:2888 –96. [45] Manzotti CN, Liu MK, Burke F, Dussably L, Zheng Y, Sansom DM. Integration of CD28 and CTLA-4 function results in differential responses of T cells to CD80 and CD86. Eur J Immunol 2006;36:1413–22. [46] Friedline RH, Brown DS, Nguyen H, Kornfeld H, Lee J, Zhang Y, et al. CD4⫹ regulatory T cells require CTLA-4 for the maintenance of systemic tolerance. J Exp Med 2009;206:421–34. [47] Wang XB, Zhao X, Giscombe R, Lefvert AK. A CTLA-4 gene polymorphism at position ⫺318 in the promoter region affects the expression of protein. Genes Immun 2002;3:233– 4. [48] Anjos S, Nguyen A, Ounissi-Benkalha H, Tessier MC, Polychronakos C. A common autoimmunity predisposing signal peptide variant of the cytotoxic Tlymphocyte antigen 4 results in inefficient glycosylation of the susceptibility allele. J Biol Chem 2002;277:46478 – 86. [49] Hegel JK, Knieke K, Kolar P, Reiner SL, Brunner-Weinzierl MC. CD152 (CTLA-4) regulates effector functions of CD8⫹ T lymphocytes by repressing eomesodermin. Eur J Immunol 2009;39:883–93.
Contents lists available at ScienceDirect
Investigation of cytotoxic T-lymphocyte-associated protein 4 gene polymorphisms in symptomatic gallstone disease Shou-Chuan Shih a,b, Horng-Woei Yang c, Tzu-Yang Chang c, Kuang-Chun Hu a,b, Shih-Chuan Chang c, Chiung-Ling Lin c, Chien-Yuan Hung a, Horng-Yuan Wang a, Marie Lin c, Yann-Jinn Lee c,d,e,* a
Department of Internal Medicine, Mackay Memorial Hospital, Taipei, Taiwan Mackay Medicine, Nursing and Management College, Taipei, Taiwan c Department of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan d Department of Pediatrics, Mackay Memorial Hospital, Taipei, Taiwan e Department of Pediatrics, Taipei Medical University, Taipei, Taiwan b
A R T I C L E
I N F O
Article history: Received 19 May 2010 Accepted 13 January 2011 Available online 25 January 2011
Keywords: CTLA4 Gallstone disease Taiwanese Single nucleotide polymorphism Case– control study
A B S T R A C T
Gallstone disease (GSD), which is increasingly prevalent in Taiwan, develops through a complex process involving genetic, environmental, and immune factors. Cytotoxic T-lymphocyte-associated protein 4 (CTLA4) limits T-cell proliferation. The present study looked for associations between symptomatic GSD and polymorphisms of the CTLA4 gene. For this case– control cross-sectional study among Taiwanese, 275 patients with symptomatic GSD and 852 controls were enrolled. Genotyping of CTLA4⫺318 C/T, +49 A/G, and CT60 A/G single nucleotide polymorphisms (SNPs) was performed by polymerase chain reaction–restriction fragment length polymorphism. The genotype, allele, carrier, and haplotype frequencies were calculated by direct counting or with Haploview 4.1 software. Genotype, allele, carrier, and haplotype frequencies of the CTLA4 SNPs studied were equally distributed in symptomatic GSD patients and controls. No significant associations between symptomatic GSD and these 3 SNPs were observed. Our data suggest that CTLA4⫺318 C/T, +49 A/G, and CT60 A/G SNPs do not confer increased susceptibility to symptomatic GSD. 䉷 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction Gallstone disease (GSD) is common in most European countries as well as in North and South America, with a prevalence of more than 10% [1– 4]. Epidemiologic surveys in Taiwan indicate a dramatic increase in the prevalence of GSD over the past 16 years from 4.3 to 10.7%, with patients presenting with acute cholecystitis, acute cholangitis, or biliary pancreatitis [5–7]. The overall prevalence of asymptomatic GSD was 5% and no significant difference in gender in a community-based study was found in Taiwan [6]. These data indicate that GSD has become the most serious gastrointestinal problem in Taiwan [8]. Gallstone biogenesis results from complicated interactions between genetic factors and high-carbohydrate, high-cholesterol, or low-fiber diets [9]. The documented risk factors of GSD include older age, female gender, high-calorie low-fiber diet, obesity, and low physical activity [10]. Recently, immune system and immunerelated genes were implicated in the gallstone formation [11–14]. Immunoglobulins, especially IgM, IgA, and IgG, not only are involved in inflammatory disorders but also contribute to the nucleation of supersaturated cholesterol in the bile and crystal formation
* Corresponding author. E-mail address: [email protected] (Dr Y.-J. Lee).
[12,13]. A murine study has also demonstrated a crucial role of T cells in GSD [14]. Another study of interleukin 4 (IL-4)-deficient mice exhibited enhanced gallstone formation in mice fed a highcholesterol diet [11]. Inflammation resulting from a series of immune responses may lead to gallstone-related symptoms [10]. In addition, inflammatory responses were involved in different types of gallstones [15,16]. However, an association between GSD and genes in the immune response pathways has not yet been conclusively demonstrated [17,18]. Given the complexity of the immune response, investigation of other immune-related genes is needed to understand genetic susceptibility to GSD. The cytotoxic T-lymphocyte-associated protein 4 gene (CTLA4) is located on chromosome 2q33 and contains 4 exons. CTLA4 is a member of the immunoglobulin superfamily that inhibits T-cell proliferation [19]. In view of the critical role of CTLA4 in regulation of the immune system [20] and the contribution of immune and inflammatory reactions to the formation of different types of gallstones [14], we hypothesized that there might be a genetic association between CTLA4 single nucleotide polymorphisms (SNPs) and GSD. We therefore analyzed 3 of the most common SNPs in the CTLA4 gene, ⫺318 C/T (rs5742909), +49 A/G (rs231775), and CT60 A/G (rs3087243), in Taiwanese patients with symptomatic GSD compared with healthy control subjects.
0198-8859/11/$32.00 - see front matter 䉷 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2011.01.004
356
S.-C. Shih et al. / Human Immunology 72 (2011) 355–358
Table 1 Genotype, allele, and carrier frequencies of CTLA4⫺318 C/T polymorphism in patients with GSD and controls Frequencies
Genotypea C/C C/T T/T Alleleb C T Carrierc C T
GSD n ⫽ 275 (%)
Control n ⫽ 852 (%)
OR (95% CI)
2
p
pc
234 (85.1) 36 (13.1) 5 (1.8)
691 (81.1) 152 (17.8) 9 (1.1)
1.33 (0.92–1.93) 0.69 (0.47–1.03) 1.73 (0.58–5.22)
2.25 3.37 0.98
0.13 0.06 0.32
0.25 0.13 0.54
504 (91.6) 46 (8.4)
1534 (90.0) 170 (10.0)
1.21 (0.86–1.71) 0.82 (0.59–1.16)
1.25 1.25
0.26 0.26
0.26 0.26
270 (98.2) 41 (14.9)
843 (98.9) 161 (18.9)
0.58 (0.19–1.74) 0.75 (0.52–1.09)
0.98 2.25
0.32 0.13
0.32 0.13
⫽ 4.19, p ⫽ 0.12 (3 ⫻ 2 contingency table). ⫽ 1.25, p ⫽ 0.26 (2 ⫻ 2 contingency table). ⫽ 1.49, p ⫽ 0.22 (2 ⫻ 2 contingency table). Data are in n (%). OR ⫽ odds ratio, 95% CI ⫽ 95% confidence interval. Hardy–Weinberg test for patients: p ⫽ 0.058; for controls: p ⫽ 0.95.
a 2
b 2 c 2
Table 2 Genotype, allele, and carrier frequencies of CTLA4 +49 A/G polymorphism in patients with GSD and controls Frequencies
Genotypea A/A A/G G/G Alleleb A G Carrierc A G
GSD n ⫽ 275 (%)
Control n ⫽ 852 (%)
OR (95% CI)
2
p
pc
25 (9.1) 122 (44.4) 128 (46.5)
89 (10.4) 381 (44.7) 382 (44.8)
0.86 (0.54–1.37) 0.99 (0.75–1.30) 1.07 (0.82–1.41)
0.42 0.01 0.25
0.52 0.92 0.62
0.77 0.99 0.86
172 (31.3) 378 (68.7)
559 (32.8) 1145 (67.2)
0.93 (0.76–1.15) 1.07 (0.87–1.32)
0.45 0.45
0.50 0.50
0.50 0.50
147 (53.5) 250 (90.9)
470 (55.2) 763 (89.6)
0.93 (0.71–1.23) 1.17 (0.73–1.86)
0.25 0.42
0.62 0.52
0.62 0.52
⫽ 0.52, p ⫽ 0.77 (3 ⫻ 2 contingency table). ⫽ 0.45, p ⫽ 0.50 (2 ⫻ 2 contingency table). c 2 ⫽ 0.15, p ⫽ 0.70 (2 ⫻ 2 contingency table). Data are in n (%). OR ⫽ odds ratio, 95% CI ⫽ 95% confidence interval. Hardy–Weinberg test for patients: p ⫽ 0.72; for controls: p ⫽ 0.75. a 2
b 2
2. Patients and methods 2.1. Patients and control subjects We enrolled 275 unrelated Taiwanese patients (125 men, 150 women) with mixed and symptomatic gallstones. GSD was diagnosed on the basis of clinical, laboratory, and imaging evidence or surgical proof. The average age at diagnosis was 52.7 ⫾ 13.0 years (23.5– 83.9 years). The control group included 852 Taiwanese (411 men, 441 women), either hospital personnel or individuals undergoing routine health examinations or minor surgery. None had a history of biliary disorders but may have had asymptomatic gallstones. Our institutional review board approved this study, and all subjects gave informed consent.
Beverly, MA), respectively. The ⫺318 C/T SNP was amplified with modified primers 5=-GTTAGGGATGCCCAGAAGAT- 3= and 5=-CTCAACTGAACAAAACAAGC-3=, resulting in a 172-bp product [23]. The +49 A/G SNP was amplified with primers 5=-AAGGCTCAGCTGAACCTGGT-3= and 5=-CTGCTGAAACAAATGAAACCC-3=, resulting in a 153-bp product [24]. The CT60 A/G SNP was amplified with primers 5=-CACCACTATTTGGGATATACC-3= and 5=-AGGTCTATATTTCAGGAAGGC-3=, resulting in a 216-bp product [25]. The individual amplified products were digested with restriction enzymes and separated on a 3.5% agarose gel (SeaKem LE agarose, Cambrex, Rockland, ME) as described in our previous study [26]. 2.5. Statistical power
dbSNP rs5742909 (⫺318 C/T) is a transition at position ⫺318 of the promoter region, rs231775 (+49 A/G) is in exon 1 at position ⫹49, and rs3087243 (CT60 A/G) at position ⫹6230 [21]. The CT60 G allele is associated with a 50% decrease in the soluble CTLA4 isoform [21]. Individuals with the ⫺318 T allele or ⫹49 G/G genotype have higher CTLA4 expression than those without these genotypes [22].
Because the asymptomatic cholelithic cases hidden in controls may influence the authenticity of our study, we adopted the feature of unselected controls built into Genetic Power Calculator software [27]. With this feature, which indicates a true random population sample (e.g., for a 1% disease, 1% of controls would also, by chance, have the disease), we designed the study to have ⬎80% power in a case– control study at a 5% significance level to detect a relative risk of 1.9 for GSD with a particular genotype and an estimated prevalence of 0.05 [6,28].
2.3. DNA extraction
2.6. Statistical analysis
DNA was extracted from fresh or frozen peripheral blood leukocytes from GSD patients and controls using standard methods.
The genotype, allele, and carrier frequencies of the ⫺318 C/T, +49 A/G, and CT60 A/G SNPs were determined by direct counting. Hardy–Weinberg equilibrium was assessed for each SNP in both patient and control groups with Haploview 4.1 software [29]. The genotype, allele, and carrier distributions of patients and controls were compared by a 2 test (3 ⫻ 2 or 2 ⫻ 2 contingency tables) with Yates’ correction where appropriate (one expected number ⬍ 5).
2.2. SNP selection
2.4. CTLA4 genotyping ⫺318 C/T, +49 A/G, and CT60 A/G SNPs were typed by polymerase chain reaction–restriction fragment length polymorphism using MseI, BstEII, and NcoI restriction enzymes (New England BioLabs,
S.-C. Shih et al. / Human Immunology 72 (2011) 355–358
357
Table 3 Genotype, allele, and carrier frequencies of CTLA4 CT60 A/G polymorphism in patients with GSD and controls Frequencies
Genotypea A/A A/G G/G Alleleb A G Carrierc A G
OR (95% CI)
2
p
pc
36 (4.2) 302 (35.5) 514 (60.3)
1.12 (0.59–2.15) 0.92 (0.69–1.22) 1.06 (0.81–1.41)
0.13 0.36 0.19
0.72 0.55 0.66
0.92 0.79 0.88
118 (21.5) 432 (78.5)
374 (21.9) 1330 (78.1)
0.97 (0.77–1.23) 1.03 (0.81–1.30)
0.06 0.06
0.81 0.81
0.81 0.81
105 (38.2) 262 (95.3)
338 (39.7) 816 (95.8)
0.94 (0.71–1.24) 0.89 (0.46–1.70)
0.19 0.13
0.66 0.72
0.66 0.72
GSD n ⫽ 275 (%)
Control n ⫽ 852 (%)
13 (4.7) 92 (33.5) 170 (61.8)
⫽ 0.43, p ⫽ 0.81 (3 ⫻ 2 contingency table). ⫽ 0.59E-01, p ⫽ 0.81 (2 ⫻ 2 contingency table). ⫽ 0.62E-01, p ⫽ 0.80 (2 ⫻ 2 contingency table). Data are in n (%). OR ⫽ odds ratio, 95% CI ⫽ 95% confidence interval. Hardy–Weinberg test for patients: p ⫽ 1.0; for controls: p ⫽ 0.37.
a 2
b 2 c 2
Table 4 Analysis of CTLA4 haplotypes in patients with GSD and controls Haplotypea
Patients 2N ⫽ 550 (%)
Controls 2N ⫽ 1074 (%)
OR (95% CI)
2
p
pc
CGG CAA TAG CAG
377.85 (68.7) 118.25 (21.5) 46.2 (8.4) 8.25 (1.5)
707.77 (65.9) 222.32 (20.7) 100.96 (9.4) 25.78 (2.4)
1.14 (0.91–1.42) 1.05 (0.82–1.35) 0.88 (0.61–1.27) 0.62 (0.28–1.37)
1.29 0.14 0.44 1.44
0.26 0.71 0.51 0.23
0.45 0.91 0.76 0.41
a p value for 4 haplotypes between patients and controls: p ⫽ 0.48 (2 ⫽ 2.49, 3 df) (4 ⫻ 2 contingency table). Haplotype inferred using Haploview 4.1, based on the ⫺318 C/T, +49 A/G, and CT60 A/G SNPs. OR ⫽ odds ratio, 95% CI ⫽ 95% confidence interval.
Odds ratios (OR) and 95% confidence intervals (95% CI) for particular genotypes, alleles, and carriers associated with the risk of GSD were calculated [30]. Frequencies of CTLA4 haplotypes (⫺318 C/T, +49 A/G, and CT60 A/G) in patients and in controls were estimated using Haploview 4.1. Linkage disequilibrium between SNPs among controls was analyzed with Haploview 4.1. Statistical differences, OR, and 95% CI for the association of various haplotypes with GSD were determined. The Bonferroni correction was used to correct for multiple comparisons where appropriate. pc values of less than 0.05 (two-tailed) were considered statistically significant. 3. Results The genotype distributions of the ⫺318 C/T, +49 A/G, and CT60 A/G SNPs in GSD patients and controls were in Hardy–Weinberg equilibrium (p ⬎ 0.4). The frequencies of genotypes, alleles, and carriers did not differ significantly between patients and controls (Tables 1–3) or by gender (data not shown). Among controls, there was strong linkage disequilibrium between ⫺318 C/T and +49 A/G (D= ⫽ 0.954, r2 ⫽ 0.207), +49 A/G and CT60 A/G (D= ⫽ 0.932, r2 ⫽ 0.5), and ⫺318 C/T and CT60 A/G (D= ⫽ 0.856, r2 ⫽ 0.023; Suppl. Fig. 1). Haplotype CGG was the most prevalent among the 4 observed. The haplotype frequencies did not differ between patients and controls (Table 4) or between men and women (data not shown). 4. Discussion Our study showed no association between the SNPs of the CTLA4 gene investigated and GSD in Taiwanese patients. There were no differences in genotype, allele, or carrier frequencies between patients with GSD and controls. A haplotype consists of a specific combination of alleles along a chromosome [31]. It contains several polymorphic sites to produce a phenotype [32–34] and is more clearly linked with a functional polymorphism than individual markers [35,36]. Therefore, haplotype-based analysis may be more powerful in detecting certain associations [36]. However, we also failed to detect a significant difference in haplotype frequencies
between patients and controls, further confirming the lack of association among these 3 CTLA4 SNPs and GSD. Mechanisms contributing to the formation of gallstones include the cholesterol and bilirubin concentrations in bile, impaired gallbladder motility, and destabilization of bile by kinetic protein factors [8,37], each of which may promote gallstone formation. Bile salt crystals and ablated gallbladder epithelial cells form proinflammatory elements that promote induction of T-cell-dependent cytokines and lead to an inflammatory response [38]. The inflammation may further interfere with gallbladder motility and alter bile emptying [38]. In the previous study, transplantation of T cells into T-cell-deficient mice significantly increased cholesterol crystals and mucin gel in the gallbladder when the mice were fed a lithogenic diet. The treated mice also had an increase in Th1-related cytokines, such as interleukin 1, interferon-␥, and tumor necrosis factor-␣ [14]. Tumor necrosis factor-␣ may induce the overproduction of MUC5AC protein and enhance mucin secretion in human gallbladder [39]. Mucin gel can agglomerate with bile salt or cholesterol crystals into gallstones. These data demonstrate that T-cell activation and Th1-related immunity are important in gallstone biogenesis. Although the pathogeneses of cholesterol and pigment gallstones are different [40,41], inflammation responses may be involved in gallstone formation of both types [15,42,43], which is why we hypothesized that genes involved in immune responses might be associated with GSD. The CTLA4 protein contains a leader sequence, a V-domain, a transmembrane domain, and a cytoplasmic tail. In conjunction with CD28, the CTLA4 protein regulates T-cell responses to maintain proper T-cell balance [23]. CTLA4 is expressed on activated T cells and then binds to B7-1 (CD80) and B7-2 (CD86) on antigenpresenting cells and transmits inhibitory signals to repress overactivation of T cells [44,45]. Ctla4-/-mice exhibit T-cell overproliferation and early death [46]. SNP -318 C/T in the promoter region alters transcription of the CTLA4 gene [47], whereas SNP ⫹49 A/G affects posttranslational modification [48], and SNP CT60 A/G limits efficient production of soluble CTLA4 mRNA [21]. If these polymor-
358
S.-C. Shih et al. / Human Immunology 72 (2011) 355–358
phisms were combined, the synergistic effect resulting in increased T-cell activity might well contribute to a variety of diseases [21]. We found no association between GSD and the 3 SNPs studied. That does not rule out the possibility that other downstream sites in the CTLA4 signaling pathway are involved in GSD susceptibility. CTLA4 regulates transcription factor FOXP3⫹ activity in the CD4⫹ pathway [46] and triggers eomesodermin, a transcription factor that activates interferon-␥ and granzyme B activity in a CD8⫹dependent manner [49]. Such signaling sites are candidates for investigation for a possible role in altering the risk of GSD. Our study may still have the possibility of a type II (false-negative) error, although this is unlikely because the study was designed to have over 80% power to detect a significant association between the SNPs studied [27]. Therefore, extensive genotyping with more SNPs, especially with haplotype tag SNPs in the CTLA4 gene, is warranted. Acknowledgments We thank Dr Mary Jeanne Buttrey for her review and criticism of our manuscript. This study was supported by Grants MMH 9362 and E-9807 from Mackay Memorial Hospital, Taipei, Taiwan. Appendix. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.humimm.2011.01.004. References [1] Borch K, Jonsson KA, Zdolsek JM, Halldestam I, Kullman E. Prevalence of gallstone disease in a Swedish population sample. Relations to occupation, childbirth, health status, life style, medications, and blood lipids. Scand J Gastroenterol 1998;33:1219 –25. [2] Everhart JE, Khare M, Hill M, Maurer KR. Prevalence and ethnic differences in gallbladder disease in the United States. Gastroenterology 1999;117:632–9. [3] Everhart JE, Yeh F, Lee ET, Hill MC, Fabsitz R, Howard BV, et al. Prevalence of gallbladder disease in American Indian populations: findings from the Strong Heart Study. Hepatology 2002;35:1507–12. [4] Shaffer EA. Gallstone disease: epidemiology of gallbladder stone disease. Best Pract Res Clin Gastroenterol 2006;20:981–96. [5] Lu SN, Chang WY, Wang LY, Hsieh MY, Chuang WL, Chen SC, et al. Risk factors for gallstones among Chinese in Taiwan. A community sonographic survey. J Clin Gastroenterol 1990;12:542– 6. [6] Chen CH, Huang MH, Yang JC, Nien CK, Etheredge GD, Yang CC, et al. Prevalence and risk factors of gallstone disease in an adult population of Taiwan: an epidemiological survey. J Gastroenterol Hepatol 2006;21:1737– 43. [7] Huang J, Chang CH, Wang JL, Kuo HK, Lin JW, Shau WY, et al. Nationwide epidemiological study of severe gallstone disease in Taiwan. BMC Gastroenterol 2009;9:63. [8] Ho KJ, Lin XZ, Yu SC, Chen JS, Wu CZ. Cholelithiasis in Taiwan. Gallstone characteristics, surgical incidence, bile lipid composition, and role of betaglucuronidase. Dig Dis Sci 1995;40:1963–73. [9] Lammert F, Wang DQ. New insights into the genetic regulation of intestinal cholesterol absorption. Gastroenterology 2005;129:718 –34. [10] Lammert F, Miquel JF. Gallstone disease: from genes to evidence-based therapy. J Hepatol 2008;48 (suppl 1):S124 –35. [11] King VL, Szilvassy SJ, Daugherty A. Interleukin-4 deficiency promotes gallstone formation. J Lipid Res 2002;43:768 –71. [12] Harvey PR, Upadhya GA, Strasberg SM. Immunoglobulins as nucleating proteins in the gallbladder bile of patients with cholesterol gallstones. J Biol Chem 1991;266:13996 – 4003. [13] Upadhya GA, Harvey PR, Strasberg SM. Effect of human biliary immunoglobulins on the nucleation of cholesterol. J Biol Chem 1993;268:5193–200. [14] Maurer KJ, Rao VP, Ge Z, Rogers AB, Oura TJ, Carey MC, et al. T-cell function is critical for murine cholesterol gallstone formation. Gastroenterology 2007; 133:1304 –15. [15] Rege RV, Prystowsky JB. Inflammatory properties of bile from dogs with pigment gallstones. Am J Surg 1996;171:197–201. [16] Rege RV, Prystowsky JB. Inflammation and a thickened mucus layer in mice with cholesterol gallstones. J Surg Res 1998;74:81–5. [17] Shih SC, Lee YJ, Liu HF, Dang CW, Chang SC, Lin SC, et al. Polymorphism in transmembrane region of MICA gene and cholelithiasis. World J Gastroenterol 2003;9:1541– 4. [18] Papasteriades C, al-Mahmoud I, Papageorgakis N, Romania S, Katsas A, Ollier W, et al. HLA antigens in Greek patients with cholelithiasis. Dis Markers 1990;8:17–21. [19] Alegre ML, Frauwirth KA, Thompson CB. T-cell regulation by CD28 and CTLA-4. Nat Rev Immunol 2001;1:220 – 8.
[20] McCoy KD, Le Gros G. The role of CTLA-4 in the regulation of T cell immune responses. Immunol Cell Biol 1999;77:1–10. [21] Ueda H, Howson JM, Esposito L, Heward J, Snook H, Chamberlain G, et al. Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease. Nature 2003;423:506 –11. [22] Ligers A, Teleshova N, Masterman T, Huang WX, Hillert J. CTLA-4 gene expression is influenced by promoter and exon 1 polymorphisms. Genes Immun 2001;2:145–52. [23] Harper K, Balzano C, Rouvier E, Mattei MG, Luciani MF, Golstein P. CTLA-4 and CD28 activated lymphocyte molecules are closely related in both mouse and human as to sequence, message expression, gene structure, and chromosomal location. J Immunol 1991;147:1037– 44. [24] Marron MP, Raffel LJ, Garchon HJ, Jacob CO, Serrano-Rios M, Martinez Larrad MT, et al. Insulin-dependent diabetes mellitus (IDDM) is associated with CTLA4 polymorphisms in multiple ethnic groups. Hum Mol Genet 1997;6: 1275– 82. [25] Torres B, Aguilar F, Franco E, Sanchez E, Sanchez-Roman J, Jimenez Alonso J, et al. Association of the CT60 marker of the CTLA4 gene with systemic lupus erythematosus. Arthritis Rheum 2004;50:2211–5. [26] Su TH, Chang TY, Lee YJ, Chen CK, Liu HF, Chu CC, et al. CTLA-4 gene and susceptibility to human papillomavirus-16-associated cervical squamous cell carcinoma in Taiwanese women. Carcinogenesis 2007;28:1237– 40. [27] Purcell S, Cherny SS, Sham PC. Genetic Power Calculator: design of linkage and association genetic mapping studies of complex traits. Bioinformatics 2003; 19:149 –50. [28] Buch S, Schafmayer C, Volzke H, Becker C, Franke A, von Eller-Eberstein H, et al. A genome-wide association scan identifies the hepatic cholesterol transporter ABCG8 as a susceptibility factor for human gallstone disease. Nat Genet 2007; 39:995–9. [29] Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 2005;21:263–5. [30] Lee YJ, Chen MR, Chang WC, Lo FS, Huang FY. A freely available statistical program for testing associations. MD Comput 1998;15:327–30. [31] International HapMap Consortium. A haplotype map of the human genome. Nature 2005;437:1299 –320. [32] Drysdale CM, McGraw DW, Stack CB, Stephens JC, Judson RS, Nandabalan K, et al. Complex promoter and coding region beta 2-adrenergic receptor haplotypes alter receptor expression and predict in vivo responsiveness. Proc Natl Acad Sci U S A 2000;97:10483– 8. [33] Joosten PH, Toepoel M, Mariman EC, Van Zoelen EJ. Promoter haplotype combinations of the platelet-derived growth factor alpha-receptor gene predispose to human neural tube defects. Nat Genet 2001;27:215–7. [34] Cowles CR, Hirschhorn JN, Altshuler D, Lander ES. Detection of regulatory variation in mouse genes. Nat Genet 2002;32:432–7. [35] Kruglyak L. The use of a genetic map of biallelic markers in linkage studies. Nat Genet 1997;17:21– 4. [36] Zaykin DV, Westfall PH, Young SS, Karnoub MA, Wagner MJ, Ehm MG. Testing association of statistically inferred haplotypes with discrete and continuous traits in samples of unrelated individuals. Hum Hered 2002;53:79 –91. [37] Paumgartner G, Sauerbruch T. Gallstones: pathogenesis. Lancet 1991;338: 1117–21. [38] van Erpecum KJ, Wang DQ, Moschetta A, Ferri D, Svelto M, Portincasa P, et al. Gallbladder histopathology during murine gallstone formation: relation to motility and concentrating function. J Lipid Res 2006;47:32– 41. [39] Finzi L, Barbu V, Burgel PR, Mergey M, Kirkwood KS, Wick EC, et al. MUC5AC, a gel-forming mucin accumulating in gallstone disease, is overproduced via an epidermal growth factor receptor pathway in the human gallbladder. Am J Pathol 2006;169:2031– 41. [40] Cahalane MJ, Neubrand MW, Carey MC. Physical– chemical pathogenesis of pigment gallstones. Semin Liver Dis 1988;8:317–28. [41] Carey MC. Pathogenesis of gallstones. Am J Surg 1993;165:410 –9. [42] Prystowsky JB, Huprikar JS, Rademaker AW, Rege RV. Human polymorphonuclear leukocyte phagocytosis of crystalline cholesterol, bilirubin, and calcium hydroxyapatite in vitro. Dig Dis Sci 1995;40:412– 8. [43] Prystowsky JB, Rege RV. The inflammatory effects of crystalline cholesterol monohydrate in the guinea pig gallbladder in vivo. Surgery 1998;123:258 – 63. [44] Manzotti CN, Tipping H, Perry LC, Mead KI, Blair PJ, Zheng Y, et al. Inhibition of human T cell proliferation by CTLA-4 utilizes CD80 and requires CD25⫹ regulatory T cells. Eur J Immunol 2002;32:2888 –96. [45] Manzotti CN, Liu MK, Burke F, Dussably L, Zheng Y, Sansom DM. Integration of CD28 and CTLA-4 function results in differential responses of T cells to CD80 and CD86. Eur J Immunol 2006;36:1413–22. [46] Friedline RH, Brown DS, Nguyen H, Kornfeld H, Lee J, Zhang Y, et al. CD4⫹ regulatory T cells require CTLA-4 for the maintenance of systemic tolerance. J Exp Med 2009;206:421–34. [47] Wang XB, Zhao X, Giscombe R, Lefvert AK. A CTLA-4 gene polymorphism at position ⫺318 in the promoter region affects the expression of protein. Genes Immun 2002;3:233– 4. [48] Anjos S, Nguyen A, Ounissi-Benkalha H, Tessier MC, Polychronakos C. A common autoimmunity predisposing signal peptide variant of the cytotoxic Tlymphocyte antigen 4 results in inefficient glycosylation of the susceptibility allele. J Biol Chem 2002;277:46478 – 86. [49] Hegel JK, Knieke K, Kolar P, Reiner SL, Brunner-Weinzierl MC. CD152 (CTLA-4) regulates effector functions of CD8⫹ T lymphocytes by repressing eomesodermin. Eur J Immunol 2009;39:883–93.