Biliary Atresia Susceptibility Research Articles

The correlation between rs2501577 gene polymorphism and biliary atresia: a systematic review and meta-analysis.

Multiple studies indicate a possible correlation between ADD3 rs2501577 and biliary atresia susceptibility; however, a conclusive determination has yet to be made. Investigate the role of ADD3 rs2501577 in biliary atresia susceptibility across diverse populations. The study protocol has been registered on PROSPERO, an international platform for systematic review registration (PROSPERO ID: CRD42023384641). The following databases will be searched until February 1, 2023: PubMed, Embase, Cochrane, CBM, Web of Science, and CNKI. Eight studies were selected from seven papers to assess the data. A total of 7651 participants were included, consisting of 1662 in the BA group and 5989 in the NC group. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed while conducting the systematic reviews and meta-analyses. Two authors independently assessed the quality of the included studies using the Newcastle-Ottawa Quality Assessment Scale. The significance of the pooled odds ratio (OR) was evaluated with a Z test, and statistical heterogeneity across studies was assessed using the I2 and Q statistics. Publication bias was assessed using Egger's and Begg's tests. The primary study outcome was the development of biliary atresia. Subgroup analysis was performed based on race, region, and assessment of Hardy-Weinberg equilibrium (HWE). The studies indicate that the ADD3 rs2501577 susceptibility locus increases the risk of developing biliary atresia, regardless of allelic, homozygote, dominant, and recessive gene inheritance models. Furthermore, ADD3 has been found to be associated with apoptosis, cell cycle, and cell damage repair based on functional analysis. The ADD3 rs2501577 polymorphic locus is associated with an increased risk of biliary atresia, particularly in Asian populations. This study recommends further investigation of the ADD3 rs2501577 locus in Asian populations to validate its role in the diagnosis of biliary atresia.

Association analysis and functional follow-up identified common variants of JAG1 accounting for risk to biliary atresia.

Background: Biliary atresia (BA) is a destructive, obliterative cholangiopathy characterized by progressive fibro-inflammatory disorder and obliteration of intra- and extrahepatic bile ducts. The Jagged1 (JAG1) gene mutations have been found in some isolated BA cases. We aim to explore the association of common variants in JAG1 with isolated BA risk in the Chinese Han population. Methods: We genotyped 31 tag single nucleotide polymorphisms covering the JAG1 gene region in 333 BA patients and 1,665 healthy controls from the Chinese population, and performed case-control association analysis. The expression patterns of JAG1 homologs were investigated in zebrafish embryos, and the roles of jag1a and jag1b in biliary development were examined by morpholino knockdown in zebrafish. Results: Single nucleotide polymorphisms rs6077861 [P Allelic = 1.74 × 10-4, odds ratio = 1.78, 95% confidence interval: 1.31-2.40] and rs3748478 (P Allelic = 5.77 × 10-4, odds ratio = 1.39, 95% confidence interval: 1.15-1.67) located in the intron region of JAG1 showed significant associations with BA susceptibility. The JAG1 homologs, jag1a and jag1b genes were expressed in the developing hepatobiliary duct of zebrafish, especially at 72 and 96h postfertilization. Knockdown of both jag1a and jag1b led to poor biliary secretion, sparse intrahepatic bile duct network and smaller or no gallbladders compared with control embryos in the zebrafish model. Conclusion: Common genetic variants of JAG1 were associated with BA susceptibility. Knockdown of JAG1 homologs led to defective intrahepatic and extrahepatic bile ducts in zebrafish. These results suggest that JAG1 might be implicated in the etiology of BA.

Exome-wide assessment of isolated biliary atresia: A report from the National Birth Defects Prevention Study using child-parent trios and a case-control design to identify novel rare variants.

The etiology of biliary atresia (BA) is unknown, but recent studies suggest a role for rare protein-altering variants (PAVs). Exome sequencing data from the National Birth Defects Prevention Study on 54 child-parent trios, one child-mother duo, and 1513 parents of children with other birth defects were analyzed. Most (91%) cases were isolated BA. We performed (1) a trio-based analysis to identify rare de novo, homozygous, and compound heterozygous PAVs and (2) a case-control analysis using a sequence kernel-based association test to identify genes enriched with rare PAVs. While we replicated previous findings on PKD1L1, our results do not suggest that recurrent de novo PAVs play important roles in BA susceptibility. In fact, our finding in NOTCH2, a disease gene associated with Alagille syndrome, highlights the difficulty in BA diagnosis. Notably, IFRD2 has been implicated in other gastrointestinal conditions and warrants additional study. Overall, our findings strengthen the hypothesis that the etiology of BA is complex.

MiR-100 rs1834306 A>G Increases Biliary Atresia Risk in Southern Han Chinese Children.

Biliary atresia (BA) is a type of severe cholestatic childhood disease that may have a genetic component. miR-100 plays a key role in regulating cell apoptosis, proliferation, and inflammatory reactions. A single-nucleotide polymorphism in miR-100 has been proven to modulate susceptibility to various diseases. We conducted a case-control retrospective study to explore the correlation between miR-100 gene polymorphism (rs1834306 A>G) and biliary atresia susceptibility in 484 Chinese patients and 1445 matched control subjects. Our results showed that rs1834306 A>G was correlated with a significantly increased risk for BA (GG vs. AA: adjusted odds ratio (OR) = 1.44, 95%confidence interval (CI) = 1.02-2.03, p = 0.041; and GG vs. AA/AG: adjusted OR = 1.39, 95%CI = 1.02-1.89, p = 0.036). Our results showed that the rs1834306 A>G polymorphism is associated with an increased risk for BA and contributes to BA susceptibility.

VEGFA rs3025039 and biliary atresia susceptibility in Chinese population: a systematic review and meta-analysis.

Previous studies have suggested an association between vascular endothelial growth factor A (VEGFA) rs3025039 polymorphism and biliary atresia (BA). However, this conclusion is controversial and there is no published pooled evidence of this association. This study was conducted and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses. The protocol was registered with PROSPERO (International Prospective Register of Systematic Reviews). A thorough search was performed on databases including PubMed, Embase, and Chinese Biomedical Database up to August 2020. This study included 846 cases of BA and 2821 controls concerning VEGFA rs3025039 polymorphism. We selected relevant studies based on the following inclusion criteria: (1) the study design was case-control and cohort and (2) the patients carried standard clinical diagnoses of BA, etc. The exclusion criteria were as follows: (1) patients with other related diseases, (2) lack of requisite information and (3) duplicate data. The OR (odd ratio) and the corresponding 95% CI (confidence interval) were calculated to estimate the association. This study on VEGFA rs3025039 polymorphism in the Chinese population included 846 cases and 2821 controls. The results showed that there was no significant association between rs3025039 and susceptibility to BA under four genetic models. The results of the subgroup analysis were similar to the overall results. This meta-analysis shows that rs3025039 was not associated with susceptibility to BA in the Chinese population. Further validation may entail additional research. CRD42020203812.

Association of common variation in ADD3 and GPC1 with biliary atresia susceptibility.

Biliary atresia (BA) is an idiopathic neonatal cholestatic disease. Recent genome-wide association study (GWAS) revealed that common variation of ADD3, GPC1, ARF6, and EFEMP1 gene was associated with BA susceptibility. We aimed to evaluate the association of these genes with BA in Chinese population. Twenty single nucleotide polymorphisms (SNPs) in these four genes were genotyped in 340 BA patients and 1,665 controls. Three SNPs in ADD3 were significantly associated with BA, and rs17095355 was the top SNP (PAllele = 3.23×10-6). Meta-analysis of published data and current data indicated that rs17095355 was associated with BA susceptibility in Asians and Caucasians. Three associated SNPs were expression quantitative trait loci (eQTL) for ADD3. Two GPC1 SNPs in high linkage disequilibrium (LD) showed nominal association with BA susceptibility (PAllele = 0.03 for rs6707262 and PAllele = 0.04 for rs6750380), and were eQTL of GPC1. Haplotype harboring these two SNPs almost reached the study-wide significance (P = 0.0035). No association for ARF6 and EFEMP1 was found with BA risk in the current population. Our study validated associations of ADD3 and GPC1 SNPs with BA risk in Chinese population and provided evidence of epistatic contributions of genetic factors to BA susceptibility.

Irrelevance of USF2 rs916145 polymorphism with the risk of biliary atresia susceptibility in Southern Chinese children.

Backgrounds: Biliary atresia (BA) is a very rare neonatal disease, however, it has been the most common cause of obstructive jaundice in infancy. The complex pathogenesis of BA is not entirely clear and a lot of possible pathogenic mechanisms have been proposed to explain the etiology of BA, including genetic, inflammatory, environmental and developmental abnormalities. As a transcription factor, USF2 gene rs916145 polymorphism has been shown to be related to the risk of BA.Methods: We examined the USF2 rs916145 genotype in a large case–control study consisting of 506 BA patients and 1473 healthy controls, using the MassARRAY iPLEX Gold system (Sequenom). Odds ratios (ORs) and 95% confidence intervals (CIs) were used to evaluate the association between the USF2 gene rs916145 polymorphism and BA susceptibility.Results: The frequency of different genotypes showed no statistical significance (GG/GC, OR: 1.09, P=0.470, 95% CI: 0.87–1.35; GG/CC, OR: 0.86, P=0.378, 95% CI: 0.62–1.20). No obvious association was revealed between the USF2 gene rs916145 polymorphism and BA susceptibility.Conclusion: USF2 rs916145 polymorphism may not be the best predictor of BA.

Epistatic Association of CD14 and NOTCH2 Genetic Polymorphisms with Biliary Atresia in a Southern Chinese Population

Biliary atresia (BA) is the most common cause of endstage liver disease in infants with poor prognosis and high mortality. The etiology of BA is still unknown, but the genetic factors have been considered as an important player in BA. We investigated the association of two cis-regulated variants in CD14 (rs2569190) and NOTCH2 (rs835576) with BA susceptibility, using the largest case-control cohort, totaling 506 BA patients and 1,473 healthy controls in a Southern Chinese population. Significant epistatic interaction between the two variants in our samples was observed (p = 8.1E−03; OR = 2.78; 95% CI: 1.32–5.88). The expression of CD14 and NOTCH2 in the BA group was consistently lower than that in the control (CC) group (0.31 ± 0.02 versus 1.00 ± 0.14; p < 0.001), which might be related to the genetic susceptibility of the genes awaiting further validation.

Association of IL18 genetic polymorphisms with increased risk of Biliary atresia susceptibility in Southern Chinese children

Biliary atresia (BA) has complex genetic etiology, characterized by different levels of hepatic fibrosis after the Kasai procedure and immune responses to the bile duct. As an activator of the two most important inflammatory cells in Biliary atresia (T cells and NK cells), IL-18 is significantly increased in BA patients. This study aims to investigate the association of Interleukin 18(IL-18) with the susceptibility to BA. We examined the association of three polymorphisms (rs549908, rs187238 and rs1946518 in IL-18) and BA susceptibility in a Southern Chinese population composed of 506 cases and 1473 controls. SNP rs187238 and rs1946518 were identified as associated with BA. Interestingly, we also observed that the intragenic synergistic epistasis between SNPs rs187238 and rs1946518 boosting the risk to BA by logistic regression and Multifactor dimensionality reduction (MDR) analysis. This study provides for the first time a direct evidence to support IL-18 as a susceptibility gene for the disease in southern Chinese children.

Association of CD14 and NOTCH2 Genetic Polymorphisms with Biliary Atresia in a Southern Chinese Population

Background: Biliary atresia (BA) is the most common cause of end-stage liver disease in infants with poor prognosis and high mortality. The etiology of BA is still unknown, but the genetic factors have been considered as an important player in BA. Methods: We investigated the association of CD14 (rs2569190) and NOTCH2 (rs835576) with BA susceptibility using the largest case-control cohort, totaling with 506 BA patients and 1473 healthy controls in a Southern Chinese population. The variants were genotyped using the MassARRAY iPLEX Gold system. The association of CD14 (rs2569190) and NOTCH2 (rs835576) with BA risk was evaluated by odds ratios (ORs) and 95% confidence intervals (CIs). The abnormal expression of CD14 and NOTCH2 in BA patients was analyzed with quantitative RT-PCR and further verified by Western blot. Results: Significant epistatic interaction between the two variants in our samples was observed (P= 8.1E-03, OR= 2.78, 95% CI: 1.32~5.88). The quantitative RT-PCR results showed that the expression of CD14 and NOTCH2 in BA group was significantly lower than that in CC group (0.31±0.02 vs. 1.00±0.14, P<0.001). Conclusions: Though the associations of CD14 (rs2569190) and NOTCH2 (rs835576) SNPs failed to replicate, the interaction between these two expression regulatory SNPs was further identified. The expression of CD14 and NOTCH2 is significantly decreased in BA patients, which might be related to the genetic susceptibility of the genes awaiting for further validation. Funding Information: This study was supported by the grant of National Natural Science Foundation of China (Grant No. 81770510, 81771629, and 81671498) and the Science and Technology Project of Guangzhou (Grant No. 201707010014). Competing Interest Declaration: The authors declare no competing financial interests. Ethical Approval Statement: This study was approved by the Institutional Review Board of Guangzhou Women and Children’s Medical Center and the experimental process is strictly abided by medical ethics. All participants have signed informed consent.

Association of polymorphism in the VEGFA gene 3'-UTR +936T/C with susceptibility to biliary atresia in a Southern Chinese Han population.

Biliary atresia (BA) is a neonatal disease characterized by chronic inflammation of the bile ducts and progressive aggravation of jaundice, but with a poor prognosis and high mortality. The etiology of BA is still uncertain which may be related to gene defect, virus infection, immune disorder, gene polymorphism. As a proinflammatory cytokine, VEGFA gene polymorphism (rs3025039) has been shown to be related to the pathogenesis of BA in Taiwanese population. We investigated the association between VEGFA gene polymorphism (rs3025039) and BA susceptibility using the largest case-control cohort, totaling with 506 BA patients and 1473 healthy controls in a Southern Chinese Han population. VEGFA gene polymorphism (rs3025039) was genotyped using the MassARRAY iPLEX Gold system (Sequenom). Odds ratios (OR) and 95% confidence intervals (CIs) were used to access the association between the VEGFA gene polymorphism (rs3025039) and BA risk. No significant association was found between the VEGFA gene polymorphism (rs3025039) and BA risk in the overall analysis. These results suggest that VEGFA gene polymorphism (rs3025039) may not be associated with the risk of BA in the Southern Chinese Han population.

Association of common genetic variants in VEGFA with biliary atresia susceptibility in Northwestern Han Chinese

Biliary atresia (BA) is a major neonatal obliterative cholangiopathy, resulting in progressive cirrhosis. The gene VEGFA encodes a heparin-binding protein that is a regulator of angiogenesis and a mediator of inflammatory reactions, and accumulating evidence have indicated that VEGFA may play a possible role in the pathogenesis of BA. Our study aim was to evaluate the association of common variants within the VEGFA gene with BA susceptibility in Northwestern Han Chinese population. Forty tag SNPs within the VEGFA gene were selected in the study, and then subsequently genotyped in 1336 Northwestern Han Chinese individuals, consisting of 311 BA patients and 1025 healthy controls. The SNP rs3025039 was found to be strongly associated with BA risk (additive P=0.000264) in our sample, and the CC genotype of rs3025039 had higher prevalence than the other two genotypes, indicating the C allele is a risk allele with an odds ratio (OR) of 1.56 and 95% confidence interval (CI) of 1.23–1.99. Haplotype analyses showed that a LD block containing rs3025039 significantly correlated with BA risk (global P<0.001). Moreover, bioinformatics analysis indicated that hsa-mir-591 and VEGFA formed miRNA/SNP target duplexes if the rs3025039 allele was in the T form, suggesting that rs3025039 may alter VEGFA expression by affecting hsa-miR-591/single-nucleotide polymorphism target duplexes. Our results indicate additional evidence supporting that there is an important role of the VEGFA gene in the increased susceptibility of BA. AbbreviationsImage 1BABiliary atresiaGWASgenome-wide association studiesSNPsingle-nucleotide polymorphismMAFminor allele frequenciesHWEHardy-Weinberg equilibriumORsOdds ratiosCIsconfidence intervalsLDlinkage disequilibriumUTRuntranslated region

Loss of a Candidate Biliary Atresia Susceptibility Gene, add3a, Causes Biliary Developmental Defects in Zebrafish.

Biliary atresia (BA) is a progressive fibroinflammatory cholangiopathy affecting the bile ducts of neonates. Although BA is the leading indication for pediatric liver transplantation, the etiology remains elusive. Adducin 3 (ADD3) and X-prolyl aminopeptidase 1 (XPNPEP1) are 2 genes previously identified in genome-wide association studies as potential BA susceptibility genes. Using zebrafish, we investigated the importance of ADD3 and XPNPEP1 in functional studies. To determine whether loss of either gene leads to biliary defects, we performed morpholino antisense oligonucleotide (MO) knockdown studies targeting add3a and xpnpep1 in zebrafish. Individuals were assessed for decreases in biliary function and the presence of biliary defects. Quantitative polymerase chain reaction was performed on pooled 5 days postfertilization larvae to assess variations in transcriptional expression of genes of interest. Although both xpnpep1 and add3a are expressed in the developing zebrafish liver, only knockdown of add3a produced intrahepatic defects and decreased biliary function. Similar results were observed in homozygous add3a mutants. MO-mediated knockdown of add3a also showed higher mRNA expression of hedgehog (Hh) targets. Inhibition of Hh signaling rescued biliary defects caused by add3a knockdown. Combined knockdown of add3a and glypican-1 (gpc1), another mediator of Hh activity that is also a BA susceptibility gene, resulted in more severe biliary defects than knockdown of either alone. Our results support previous studies identifying ADD3 as a putative genetic risk factor for BA susceptibility. Our results also provide evidence that add3a may be affecting the Hh pathway, an important factor in BA pathogenesis.

761 Zebrafish Studies Show That Add3, Not Xpnpep1, Is a Biliary Atresia Risk Gene

Biliary atresia (BA) is a progressive fibro-inflammatory cholangiopathy affecting the intraand extrahepatic bile ducts of neonates. Although BA is the most common identifiable cause of obstructive jaundice in infants, and is the leading indication for pediatric liver transplantation, the etiology remains elusive. We investigated the importance of genes identified in genome-wide association studies (GWAS) of BA patients using zebrafish. The Matthews's lab has demonstrated the utility of the zebrafish system to test the functionality of genes identified in GWAS of BA by demonstrating that knockdown of gpc1 leads to biliary defects. A prior GWAS examined single-nucleotide polymorphisms in 200 Han Chinese BA patients and 481 ethnically matched controls. The strongest association was found for a region located between the XPNPEP1 and ADD3 genes on 10q24.2. Our genetic analysis confirmed the importance of this region in a separate cohort of patients. To determine whether loss of xpnpep1 and/or add3a leads to biliary defects we performed knockdown studies of the respective genes using morpholino antisense oligonucleotides (MO). We then examined their biliary function using the lipid reporter PED6, biliary development using cytokeratin immunostaining, and gene expression using quantitative PCR. We confirmed that xpnpep1 and add3a are expressed in the developing zebrafish liver. Knockdown of add3a led to decreased biliary function by PED6 screening, while xpnpep1 knockdown had only a mild effect. There were developmental biliary defects in the add3a morphants, as demonstrated by cytokeratin immunostaining. The transcription factor vhnf1, implicated in biliary development, was significantly downregulated in add3a morphants but not xpnpep1 morphants. add3a morphants also demonstrated increased expression of gli2a, a Hedgehog target, which is consistent with prior studies of BA patients and of zebrafish models of BA such as gpc1 knockdown. Interestingly, knockdown of both add3a and gpc1 resulted in a synergistic disruptive effect on biliary development. While GWAS identified ADD3 and XPNPEP1 as potential BA susceptibility genes, our results suggest that ADD3 is likely the more important gene in this regard. Like gpc1, add3a acts via Hedgehog signaling, supporting a role for this important pathway in BA pathogenesis.

762 Control of Microenvironmental Cues Enables the Differentiation of Pluripotent Stem Cell Derived Hepatocyte-Like Cells to an Adult Phenotype

Biliary atresia (BA) is a progressive fibro-inflammatory cholangiopathy affecting the intraand extrahepatic bile ducts of neonates. Although BA is the most common identifiable cause of obstructive jaundice in infants, and is the leading indication for pediatric liver transplantation, the etiology remains elusive. We investigated the importance of genes identified in genome-wide association studies (GWAS) of BA patients using zebrafish. The Matthews's lab has demonstrated the utility of the zebrafish system to test the functionality of genes identified in GWAS of BA by demonstrating that knockdown of gpc1 leads to biliary defects. A prior GWAS examined single-nucleotide polymorphisms in 200 Han Chinese BA patients and 481 ethnically matched controls. The strongest association was found for a region located between the XPNPEP1 and ADD3 genes on 10q24.2. Our genetic analysis confirmed the importance of this region in a separate cohort of patients. To determine whether loss of xpnpep1 and/or add3a leads to biliary defects we performed knockdown studies of the respective genes using morpholino antisense oligonucleotides (MO). We then examined their biliary function using the lipid reporter PED6, biliary development using cytokeratin immunostaining, and gene expression using quantitative PCR. We confirmed that xpnpep1 and add3a are expressed in the developing zebrafish liver. Knockdown of add3a led to decreased biliary function by PED6 screening, while xpnpep1 knockdown had only a mild effect. There were developmental biliary defects in the add3a morphants, as demonstrated by cytokeratin immunostaining. The transcription factor vhnf1, implicated in biliary development, was significantly downregulated in add3a morphants but not xpnpep1 morphants. add3a morphants also demonstrated increased expression of gli2a, a Hedgehog target, which is consistent with prior studies of BA patients and of zebrafish models of BA such as gpc1 knockdown. Interestingly, knockdown of both add3a and gpc1 resulted in a synergistic disruptive effect on biliary development. While GWAS identified ADD3 and XPNPEP1 as potential BA susceptibility genes, our results suggest that ADD3 is likely the more important gene in this regard. Like gpc1, add3a acts via Hedgehog signaling, supporting a role for this important pathway in BA pathogenesis.

O56 SOFOSBUVIR/LEDIPASVIR WITH AND WITHOUT RIBAVIRIN FOR 8 WEEKS COMPARED TO SOFOSBUVIR/LEDIPASVIR FOR 12 WEEKS IN TREATMENT-NAÏVE NON-CIRRHOTIC GENOTYPE-1 HCV-INFECTED PATIENTS: THE PHASE 3 ION-3 STUDY

Biliary atresia (BA) is a progressive fibro-inflammatory cholangiopathy affecting the intraand extrahepatic bile ducts of neonates. Although BA is the most common identifiable cause of obstructive jaundice in infants, and is the leading indication for pediatric liver transplantation, the etiology remains elusive. We investigated the importance of genes identified in genome-wide association studies (GWAS) of BA patients using zebrafish. The Matthews's lab has demonstrated the utility of the zebrafish system to test the functionality of genes identified in GWAS of BA by demonstrating that knockdown of gpc1 leads to biliary defects. A prior GWAS examined single-nucleotide polymorphisms in 200 Han Chinese BA patients and 481 ethnically matched controls. The strongest association was found for a region located between the XPNPEP1 and ADD3 genes on 10q24.2. Our genetic analysis confirmed the importance of this region in a separate cohort of patients. To determine whether loss of xpnpep1 and/or add3a leads to biliary defects we performed knockdown studies of the respective genes using morpholino antisense oligonucleotides (MO). We then examined their biliary function using the lipid reporter PED6, biliary development using cytokeratin immunostaining, and gene expression using quantitative PCR. We confirmed that xpnpep1 and add3a are expressed in the developing zebrafish liver. Knockdown of add3a led to decreased biliary function by PED6 screening, while xpnpep1 knockdown had only a mild effect. There were developmental biliary defects in the add3a morphants, as demonstrated by cytokeratin immunostaining. The transcription factor vhnf1, implicated in biliary development, was significantly downregulated in add3a morphants but not xpnpep1 morphants. add3a morphants also demonstrated increased expression of gli2a, a Hedgehog target, which is consistent with prior studies of BA patients and of zebrafish models of BA such as gpc1 knockdown. Interestingly, knockdown of both add3a and gpc1 resulted in a synergistic disruptive effect on biliary development. While GWAS identified ADD3 and XPNPEP1 as potential BA susceptibility genes, our results suggest that ADD3 is likely the more important gene in this regard. Like gpc1, add3a acts via Hedgehog signaling, supporting a role for this important pathway in BA pathogenesis.

P1163 ALL-ORAL THERAPY WITH DACLATASVIR IN COMBINATION WITH ASUNAPREVIR AND BMS-791325 FOR TREATMENT-NAIVE PATIENTS WITH CHRONIC HCV GENOTYPE 4 INFECTION

Biliary atresia (BA) is a progressive fibro-inflammatory cholangiopathy affecting the intraand extrahepatic bile ducts of neonates. Although BA is the most common identifiable cause of obstructive jaundice in infants, and is the leading indication for pediatric liver transplantation, the etiology remains elusive. We investigated the importance of genes identified in genome-wide association studies (GWAS) of BA patients using zebrafish. The Matthews's lab has demonstrated the utility of the zebrafish system to test the functionality of genes identified in GWAS of BA by demonstrating that knockdown of gpc1 leads to biliary defects. A prior GWAS examined single-nucleotide polymorphisms in 200 Han Chinese BA patients and 481 ethnically matched controls. The strongest association was found for a region located between the XPNPEP1 and ADD3 genes on 10q24.2. Our genetic analysis confirmed the importance of this region in a separate cohort of patients. To determine whether loss of xpnpep1 and/or add3a leads to biliary defects we performed knockdown studies of the respective genes using morpholino antisense oligonucleotides (MO). We then examined their biliary function using the lipid reporter PED6, biliary development using cytokeratin immunostaining, and gene expression using quantitative PCR. We confirmed that xpnpep1 and add3a are expressed in the developing zebrafish liver. Knockdown of add3a led to decreased biliary function by PED6 screening, while xpnpep1 knockdown had only a mild effect. There were developmental biliary defects in the add3a morphants, as demonstrated by cytokeratin immunostaining. The transcription factor vhnf1, implicated in biliary development, was significantly downregulated in add3a morphants but not xpnpep1 morphants. add3a morphants also demonstrated increased expression of gli2a, a Hedgehog target, which is consistent with prior studies of BA patients and of zebrafish models of BA such as gpc1 knockdown. Interestingly, knockdown of both add3a and gpc1 resulted in a synergistic disruptive effect on biliary development. While GWAS identified ADD3 and XPNPEP1 as potential BA susceptibility genes, our results suggest that ADD3 is likely the more important gene in this regard. Like gpc1, add3a acts via Hedgehog signaling, supporting a role for this important pathway in BA pathogenesis.

Evidence From Human and Zebrafish That GPC1 Is a Biliary Atresia Susceptibility Gene

Biliary atresia (BA) is a progressive fibroinflammatory disorder of infants involving the extrahepatic and intrahepatic biliary tree. Its etiology is unclear but is believed to involve exposure of a genetically susceptible individual to certain environmental factors. BA occurs exclusively in the neonatal liver, so variants of genes expressed during hepatobiliary development could affect susceptibility. Genome-wide association studies previously identified a potential region of interest at 2q37. We continued these studies to narrow the region and identify BA susceptibility genes. We searched for copy number variants that were increased among patients with BA (n = 61) compared with healthy individuals (controls; n = 5088). After identifying a candidate gene, we investigated expression patterns of orthologues in zebrafish liver and the effects of reducing expression, with morpholino antisense oligonucleotides, on biliary development, gene expression, and signal transduction. We observed a statistically significant increase in deletions at 2q37.3 in patients with BA that resulted in deletion of one copy of GPC1, which encodes glypican 1, a heparan sulfate proteoglycan that regulates Hedgehog signaling and inflammation. Knockdown of gpc1 in zebrafish led to developmental biliary defects. Exposure of the gpc1 morphants to cyclopamine, a Hedgehog antagonist, partially rescued the gpc1-knockdown phenotype. Injection of zebrafish with recombinant Sonic Hedgehog led to biliary defects similar to those of the gpc1 morphants. Liver samples from patients with BA had reduced levels of apical GPC1 in cholangiocytes compared with samples from controls. Based on genetic analysis of patients with BA and zebrafish, GPC1 appears to be a BA susceptibility gene. These findings also support a role for Hedgehog signaling in the pathogenesis of BA.

+276 G/T single nucleotide polymorphism of the adiponectin gene is associated with the susceptibility to biliary atresia

Biliary atresia (BA) is an intractable neonatal inflammatory and obliterative cholangiopathy, leading to progressive fibrosis and cirrhosis. Adiponectin, an anti-inflammatory adipokine, is known to play a possible role in liver diseases. The objective of our study was to determine the relationship between adiponectin gene polymorphisms and BA susceptibility. A total of 106 BA patients and 107 healthy controls were included in this study. Two single nucleotide polymorphisms (SNPs) of the adiponectin gene, +45T/G (rs2241766) and +276G/T (rs1501299), were genotyped by polymerase chain reaction-restriction fragment length polymorphism analysis. Genotype distributions of +45 T/G and +276 G/T SNPs were seen in the Hardy-Weinberg equilibrium for both BA patients and controls. The frequency of the G/G genotype at +276G/T was significantly higher in BA patients than in the controls (P=0.009). Regarding +45T/ G in BA patients, the frequency of the T/T genotype tended to be lower than in the controls, but the difference was not significant. Moreover, the G allele at +276G/T in BA patients was more common than in the controls (P=0.0043). In contrast, the frequency of the T allele at +45T/G was not significantly different between BA patients and the controls. None of the haplotypes studied was found to significantly influence the risk of BA. +276G/T SNP is strongly associated with BA, particularly with the G allele. We postulate that the +276G/T adiponectin gene polymorphism confers increased susceptibility to BA.

Genetic variability of interleukin4 gene in Taiwanese children with biliary atresia

Biliary atresia (BA) is a neonatal cholangiopathy of unknown etiology that leads to biliary cirrhosis and is the most common cause of liver transplantation in children. A still undetermined hepatobiliary viral infection may elicit an uncontrollable autoimmune response against the biliary epithelial cells in genetically predisposed children and culminates in atresia of the biliary trees. Interleukin 4 (IL4) is crucial for the differentiation of naive T helper cells into the T helper 2 effector cells that promote humoral immunity. This study aims to investigate whether polymorphisms of the IL4 gene are associated with susceptibility to BA. Genomic DNA was extracted from whole blood samples of 53 Taiwanese children with BA and 904 ethnically-matched healthy controls. The IL4 –590 C/T, –33 C/T, and 8375 A/G polymorphisms were genotyped using the Pre-Developed TaqMan Allelic Discrimination Assay in a real-time polymerase chain reaction system. No significant difference between children with BA and healthy controls were found when comparing genotype, allele, carrier, and haplotype frequencies of these IL4 gene variants. These results suggest that the tested polymorphisms of IL4 gene are unlikely to contribute significantly to BA susceptibility in Taiwanese children.