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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