Journal of Pediatric Gastroenterology & Nutrition:
Original Articles: Hepatology and Nutrition
Association of Polymorphisms in the Interleukin-18 Gene With Susceptibility to Biliary Atresia
Lee, Hung-Chang*,‡; Chang, Tzu-Yang†; Yeung, Chun-Yan*,§; Chan, Wai-Tao*; Jiang, Chuen-Bin*; Chan, Hui-Wen†; Chen, Wei-Fang†; Yang, Horng-Woei†; Lin, Marie†; Lee, Yann-Jinn*,†,‡
*Departments of Pediatrics, Taiwan
†Medical Research, Mackay Memorial Hospital, Taiwan
‡Department of Pediatrics, Taipei Medical University, Taiwan
§Mackay Medicine, Nursing, and Management College, Taipei, Taiwan.
Received 6 December, 2010
Accepted 16 January, 2011
Address correspondence and reprint requests to Yann-Jinn Lee, MD, Department of Pediatrics, Mackay Memorial Hospital, No. 45 Min-Sheng Rd, Tamshui 25115, Taipei County, Taiwan (e-mail: firstname.lastname@example.org).
This research was supported by grants MMH 9780 and MMH E-98007 from Mackay Memorial Hospital, Taiwan.
The authors report no conflicts of interest.
Background and Objective: Biliary atresia (BA) is a destructive inflammatory obliterative cholangiopathy of neonates that affects both intrahepatic and extrahepatic bile ducts. Although the etiology is unknown, immunologically mediated injury of the bile ducts triggered by as yet unidentified infectious agents is likely to play a critical role. Interleukin-18 (IL-18) is a proinflammatory cytokine that plays an important role in immune, infectious, and inflammatory diseases because of its induction of interferon-gamma. In this study, we investigated whether polymorphisms of the IL18 gene were associated with susceptibility to BA.
Patients and Methods: Genomic DNA was extracted from whole-blood samples of 50 Taiwanese children with BA and 1117 ethnically matched healthy controls. The IL18 –1297 T/C, –607 C/A, –137 G/C, and +105 A/C polymorphisms were genotyped using the TaqMan assay.
Results: No statistically significant differences of genotype, allele, carrier, and haplotype frequencies of these IL18 gene variants were found between children with BA and healthy controls.
Conclusions: Our data suggest that the IL18 gene does not play a major role in BA predisposition in Taiwanese children.
Biliary atresia (BA) is the most common and important neonatal hepatobiliary disorder. It is characterized by fibrotic obliteration of the extrahepatic bile duct and intrahepatic periductal inflammation and injury. Although prompt Kasai operation is effective in improving bile drainage, progressive inflammation and fibrosis of intrahepatic bile ducts lead to biliary cirrhosis in the majority of patients. At that point, liver transplantation is the only hope for long-term survival. With a better understanding of the pathogenetic mechanisms of BA, novel nontransplant therapies could be developed to address the disease. A theory that BA is caused by a specific perinatal hepatobiliary viral infection and secondary generation of an autoimmune-mediated bile duct injury has been proposed (1). Histologic examination of affected bile ducts has revealed an exclusively T-cell–mediated inflammatory response (2–5). A number of other studies have shown that the portal tracts of patients with BA are laden with macrophages (5–8). These findings support that BA is an immune-mediated inflammatory disease.
The pleiotropic cytokine interleukin-18 (IL-18) is a member of the IL-1 family and was originally described as an inducer of interferon-gamma (IFN-γ) production (9). It is predominantly secreted by activated Kupffer cells, macrophages, and dendritic cells (9,10), and participates in both innate and acquired immunity. The major functions of IL-18 include production of IFN-γ from activated T lymphocytes and natural killer (NK) cells (9), enhancement of T- and NK-cell maturation (11), induction of proinflammatory cytokines and chemokines (12,13), and augmentation of Fas ligand (FasL)–mediated and perforin-dependent cytotoxicity of T and NK cells (14–16). Serum IL-18 levels have been reported to increase and correlate with severity in various autoimmune and inflammatory diseases (17,18), including BA (8). These findings imply that IL-18 may be involved in the pathogenesis of BA.
The human IL-18 gene (IL18) is located on chromosome 11q22.2-q22.3 (19) and contains 6 exons and 5 introns. A number of single nucleotide polymorphisms (SNPs) in the IL18 gene have been associated with differential levels of gene transcription and protein production. For example, –1297 T/C, –607 C/A, and –137 G/C SNPs in the promoter region cause differences in transcription factor binding and have an impact on IL18 gene activity (20,21). The +105 A/C polymorphism in exon 4 affects IL-18 production by purified peripheral blood monocytes (22). Some of these polymorphisms have therefore been investigated in several autoimmune and chronic inflammatory diseases (23); however, no report has been published on IL18 gene polymorphisms for the risk of BA. The aim of the present study was to examine whether specific IL18 gene SNPs, including –1297 T/C, –607 C/A, –137 G/C, and +105 A/C, and their haplotypes are associated with BA.
PATIENTS AND METHODS
From 1981 to 2006, 211 unrelated Taiwanese children were diagnosed at Mackay Memorial Hospital as having BA, of whom 33 died and 23 were lost to follow-up. The parents of the remaining 155 were contacted. Parental consent was given for 50 children (24 boys and 26 girls) to be enrolled as the study group. Their ages were 58.3 ± 28.3 (mean ± SD) (range 3–191) days at Kasai operation, 4.8 ± 4.5 (0.3–21.6) years at blood sampling, and 10.0 ± 5.4 (0.9–28.0) years at the most recent follow-up. The diagnosis of BA was based on preoperative laboratory and imaging studies and confirmed by exploratory laparotomy with operative cholangiography. Pathological examination of the liver and extrahepatic biliary tree after Kasai surgery further confirmed the diagnosis. Seven (14%) patients had progressive hepatic failure and were treated with liver transplantation. None of the patients had any associated congenital malformations.
A group of 1117 (395 boys and 722 girls) unrelated, healthy, Taiwanese subjects served as a control group. None had any history of BA, autoimmune or liver disease, or underwent liver transplantation. Informed written consent was obtained from all of the participants for use of their blood for the study. The study design conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the institutional review board of Mackay Memorial Hospital.
Genomic DNA was extracted from peripheral blood samples using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). The IL18 –1297 T/C (dbSNP ID: rs360719), –607 C/A (dbSNP ID: rs1946518), –137 G/C (dbSNP ID: rs187238), and +105 A/C (dbSNP ID: rs549908) genotypes were determined using the TaqMan assay (Applied Biosystems, Foster City, CA). Briefly, polymerase chain reactions (PCR) were carried out in a 96-well GeneAmp PCR System 9700 (Applied Biosystems) with 10 ng of genomic DNA, 5 μL of TaqMan Universal PCR Master Mix, 0.5 μL of 20 × Assay Mix, and ddH2O to a final volume of 10 μL. Thermal cycle conditions were as follows: denaturation at 95°C for 10 minutes, followed by 40 cycles of denaturation at 92°C for 15 seconds, and annealing and extension at 60°C for 1 minute. After PCR, the TaqMan assay plates were transferred to an ABI PRISM 7000 Sequence Detection System (Applied Biosystems) in which the endpoint fluorescence intensity in each well of the plate was read. The allele-specific fluorescence data from each plate were analyzed using SDS version 1.1 software (Applied Biosystems) to automatically determine the genotype of each sample.
Genotype, allele, and carrier frequencies of the IL18 –1297 T/C, –607 C/A, –137 G/C, and +105 A/C SNPs were determined by direct counting. The Hardy-Weinberg equilibrium was assessed for each SNP in both the control and study groups by chi square analysis. The frequencies of IL18 haplotypes and linkage disequilibrium (LD) between paired SNPs in controls and children with BA were estimated using Haploview 4.1 and PyPop 0.7.0 programs (24,25). Haplotypes with frequencies of <5% were grouped together. Statistical differences in genotype, allele, carrier, and haplotype distributions between controls and children with BA were performed using the chi square test with Yates correction where appropriate (1 expected number <5). Odds ratios and 95% confidence intervals were also calculated (26). Bonferroni correction was used for multiple comparisons where appropriate. Two-tailed corrected P (Pc) values < 0.05 were considered to be statistically significant.
Before the study, the statistical power necessary to detect an effect of an IL18 SNP on susceptibility to BA was determined using the Genetic Power Calculator (27). The sample size was determined based on detecting a relative risk for BA of 2.5 for each SNP with an estimated prevalence of 120/100,000 (28), with a power of >80% at a significance level of 5%.
All of the investigated polymorphisms of the IL18 gene were successfully genotyped in 1117 healthy controls and 50 children with BA. The genotype, allele, and carrier frequencies of the –1297 T/C, –607 C/A, –137 G/C, and +105 A/C SNPs in the controls and children with BA are summarized in Tables 1 to 4. For all SNPs, the distribution of genotypes did not deviate from Hardy-Weinberg equilibrium in both control and study groups (Tables 1–4). After comparing the children with BA and the controls, we found no statistically significant deviation in the distribution of genotypes, alleles, or carriers for any of these genetic variants. LD analysis for all SNP pairs revealed that there were strong pairwise LD coefficients (D′) among all SNPs (D′ 0.96–1.00, P < 0.00001 in controls, D′ 0.92–1.00, P < 0.00001 in patients), indicating that the SNPs were highly associated with each other and that they were all part of the same LD block.
We also analyzed the possible haplotypes constructed by the 4 SNPs in the controls and children with BA (Table 5). From this analysis, 3 common haplotypes (frequency > 5%) were predicted and the most common haplotype was –1297 T/–607 C/–137 G/+105 A (TCGA) with a frequency of 48.9% in controls and 50% in children with BA. No statistically significant differences were found in the distribution of haplotypes between the controls and children with BA.
In this study, we investigated the distributions of 4 functional polymorphisms of the IL18 gene in a Taiwanese population and studied their correlation with BA. The data obtained show that no significant differences in genotype, allele, carrier, and haplotype frequency between children with BA and controls were found concerning any of the studied IL18 gene SNPs. Our study therefore implies that none of the polymorphisms investigated is likely to have a major effect on BA susceptibility.
The etiology of BA is unknown, but inflammation-induced apoptosis of biliary epithelial cells (BECs) has been considered to play an important role in the development of BA. The hypothesis that apoptosis is a potential pathogenic mechanism of BA was first suggested by a report of significantly increased apoptotic cells in intrahepatic bile ducts of affected infants (29). An investigation of the expression of apoptotic molecules in children with BA revealed an overexpression of FasL in BECs that also displayed markers of apoptosis (30). Similar findings were found in different studies, which reported that another apoptosis promoter, tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), stained positive for BECs (31), and enhancements of TRAIL expression and apoptosis were found in BECs of patients with BA (32). A recent study using the rotavirus-induced murine model of BA found that cellular apoptosis was observed in intrahepatic and extrahepatic bile ducts (33). The same group further substantiated that the induction of apoptosis in BECs was attributable to the synergy between IFN-γ and tumor necrosis factor-alpha. Collectively, these data suggest that biliary apoptosis is associated with the pathogenesis of BA.
The Fas/FasL system is involved in the induction of apoptosis, and IL-18 has been reported to enhance FasL expression and apoptosis in Fas-bearing cells. An in vitro study revealed that NK cells treated with IL-18 expressed greater amounts of FasL and showed a greater capacity for killing FasL-sensitive target cells (14). A similar study also found that IL-18 can enhance the FasL-mediated cytotoxicity of CD4+ T helper 1 cells (15). In addition, FasL expression on CD8+ T cells has been shown to increase after IFN-γ treatment (34). This suggests that IL-18 may augment the FasL expression on CD8+ T cells via IFN-γ induction. Another cell death pathway is triggered by the interaction of TRAIL with TRAIL-sensitive cells. It has been reported that IFN-γ can induce TRAIL expression on liver NK cells and macrophages (35,36). Therefore, IL-18 may be responsible for the TRAIL expression on liver NK cells and macrophages. Because simultaneous exposure of BECs to IFN-γ and tumor necrosis factor-alpha induces apoptosis (33), IL-18 may constitute a key trigger of apoptosis in BECs by means of IFN-γ induction. Given the constitutive expression of apoptosis-inducing receptors (Fas, TRAIL-DR4, and TRAIL-DR5) in BECs (31,32,37), the enriched population of infiltrates found in the bile ducts of BA specimens (CD4+, CD8+ T cells, NK cells, and macrophages) (38,39), and the increased serum IL-18 levels in patients with BA (8), it is conceivable to reason that IL-18 may be involved in the activation of apoptosis in BECs and the ultimate fibro-obliteration of biliary trees in cases of BA. We were therefore surprised to find that there were no differences in the distribution of genotypes, alleles, carriers, and haplotypes between controls and children with BA for the polymorphisms we studied.
These results could have arisen from a type II statistical error (false-negative results). Because we powered the study to detect an allelic relative risk of at least 2.5, we may have missed a smaller effect that would have been evident had the sample size been larger. Another possible explanation for the findings is that the polymorphisms selected in our study do not cover the gene completely and extensively. Furthermore, a more influential role played by IL-18 in disease progression rather than being a risk factor conferring increased susceptibility to BA may account for our negative findings.
In conclusion, this study did not provide evidence to support associations between the 4 SNPs of the IL18 gene and BA in Taiwanese children. Our data suggest that this gene is not a major susceptibility gene for BA in our population. It is necessary, however, to validate or replicate these results in other independent large-size groups or other ethnic populations.
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