Pathogenesis Of Biliary Atresia Research Articles

Human poliovirus receptor contributes to biliary atresia pathogenesis by exacerbating natural-killer-cell-mediated bile duct injury.

Natural killer (NK) cells play an important role in biliary atresia (BA) pathogenesis; human poliovirus receptor (PVR) is an important NK-cell modulator. Here, we explored the role of PVR in BA pathogenesis. Poliovirus receptor expression and NK cell-associated genes were detected in human BA samples and a rotavirus-induced BA mouse model using quantitative PCR and immunofluorescence staining. Chemically modified small interfering RNA silenced PVR expression in the BA model, and its effects on the population and function of intrahepatic NK cells were investigated using flow cytometry (FCM). The effects of PVR overexpression and knockdown on proliferation, apoptosis and NK-cell-mediated lysis of cultured human cholangiocytes were analysed using FCM and cell viability assays. Serum PVR, high-mobility group box 1 (HMGB1), and interleukin-1beta (IL-1beta) levels were measured in a cohort of 50 patients using ELISA. Poliovirus receptor expression was upregulated in the biliary epithelium of BA patients and BA model and was positively correlated with the population and activation of intrahepatic NK cells. Silencing of PVR expression impaired the cytotoxicity of NK cells, reduced inflammation and protected mice from rotavirus-induced BA. Activation of the TLR3-IRF3 signalling pathway induced PVR expression in cultured cholangiocytes. PVR overexpression promoted proliferation and inhibited the apoptosis of cholangiocytes but exacerbated NK cell-mediated cholangiocyte lysis. Serum PVR levels were elevated in BA patients and were positively correlated with HMGB1 and IL-1beta levels. Poliovirus receptor contributes to BA pathogenesis by regulating NK cell-mediated bile duct injury; PVR has the value as a biomarker of BA.

Beta-amyloid Deposition in Biliary Atresia Reduces Liver Regeneration by Inhibiting Energy Metabolism and Mammalian Target of Rapamycin Signaling.

Biliary atresia (BA) is a devastating obstructive bile duct disease found in newborns. This study aims to investigate the roles and involved mechanisms of beta-amyloid (Aβ) in the pathogenesis of BA. We examined the distribution of Aβ protein and its precursor in the livers of patients with BA. A murine liver organoid and a zebrafish model were established to investigate the exact roles of Aβ in liver regeneration for BA. Both Aβ mRNA and protein significantly increased in livers of infants with BA and deposited around the central vein. In the plasma, Aβ elevated significantly in patients with BA and positively correlated with liver injury progression. In vitro , Aβ treatment induced abnormal morphology and caused impaired growth in liver organoids. Energy metabolism analysis demonstrated Aβ increased aerobic glycolysis and reduced ATP synthase in organoids, in which the mammalian target of rapamycin signaling was suppressed. In vivo , Aβ42 exposure caused liver degeneration in zebrafish larvae. Aβ depositing in livers of infants with BA reduced the liver regeneration through attenuating mitochondrial respiration and mammalian target of rapamycin signaling.

Identification of signature of gene expression in biliary atresia using weighted gene co-expression network analysis.

Biliary atresia (BA) is the most common cause of obstructive jaundice during the neonatal period. This study aimed to identify gene expression signature in BA. The datasets were obtained from the Gene Expression Omnibus database. Weighted gene co-expression network analysis identified a critical module associated with BA, whereas Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed the functions of the essential modules. The high-connectivity genes in the most relevant module constructed protein–protein interaction networks via the string website and Cytoscape software. Hub genes screened by lasso regression consisted of a disease classification model using the randomforest method. Receiver operating characteristic curves were used to assess models’ sensitivity and specificity and the model was verified using the internal and external validation sets. Ten gene modules were constructed by WGCNA, of which the brown module had a strong positive correlation with BA, comprising 443 genes. Functional enrichment analysis revealed that module genes were mainly involved in biological processes, such as extracellular matrix organization, cell adhesion, inflammatory response, and the Notch pathway (P < .001), whereas these genes were involved in the metabolic pathways and cell adhesion molecules (P < .001). Thirty-nine high-connectivity genes in the brown module constructed protein-protein interaction networks. keratin 7 (KRT7) and C-X-C motif chemokine ligand 8 (CXCL8) were used to construct a diagnostic model that had an accuracy of 93.6% and the area under the receiver operating curves for the model was 0.93. The study provided insight into the signature of gene expression and possible pathogenesis of BA; furthermore, it identified that the combination of KRT7 and CXCL8 could be a potential diagnostic model for BA.

HIF-1alpha-pathway activation in cholangiocytes of patients with biliary atresia: An immunohistochemical/molecular exploratory study

BackgroundBiliary atresia is a neonatal disease characterized by choledochal obstruction and progressive cholangiopathy requiring liver transplantation in most patients. Hypoxia-ischemia affecting the biliary epithelium may lead to biliary obstruction. We hypothesized that ischemic cholangiopathy involving disruption of the peribiliary vascular plexus could act as a triggering event in biliary atresia pathogenesis. MethodsLiver and porta hepatis paraffin-embedded samples of patients with biliary atresia or intrahepatic neonatal cholestasis (controls) were immunohistochemically evaluated for HIF-1alpha-nuclear signals. Frozen histological samples were analyzed for gene expression in molecular profiles associated with hypoxia-ischemia. Prospective clinical-laboratory and histopathological data of biliary atresia patients and controls were reviewed. ResultsImmunohistochemical HIF-1alpha signals localized to cholangiocytes were detected exclusively in liver specimens from biliary atresia patients. In 37.5% of liver specimens, HIF-1alpha signals were observed in biliary structures involving progenitor cell niches and peribiliary vascular plexus. HIF-1alpha signals were also detected in biliary remnants of 81.8% of porta hepatis specimens. Increased gene expression of molecules linked to REDOX status, biliary proliferation, and angiogenesis was identified in biliary atresia liver specimens. In addition, there was a trend towards decreased GSR expression levels in the HIF-1alpha-positive group compared to the HIF-1alpha-negative group. ConclusionActivation of the HIF-1alpha pathway may be associated with the pathogenesis of biliary atresia, and additional studies are necessary to confirm the significance of this finding. Ischemic cholangiopathy and REDOX status disturbance are putative explanations for HIF-1alpha activation. These findings may give rise to novel lines of clinical and therapeutic investigation in the BA field.

MRNA and lncRNA expression profiles of liver tissues in children with biliary atresia

Progressive liver fibrosis is the most common phenotype in biliary atresia (BA). A number of pathways contribute to the fibrosis process so comprehensive understanding the mechanisms of liver fibrosis in BA will pave the way to improve patient's outcome after operation. In this study, the differentially expressed profiles of mRNAs and long non-coding RNAs from BA and choledochal cyst (CC) liver tissues were investigated and analyzed, which may provide potential clues to clarify hepatofibrosis mechanism in BA. A total of two BA and two CC liver tissue specimens were collected, the expression level of mRNAs and lncRNAs was detected by RNA sequencing. Differentially expressed mRNAs (DEmRNAs) were functionally annotated and protein-protein interaction networks (PPI) was established to predict the biological roles and interactive relationships. Differentially expressed lncRNAs (DElncRNAs) nearby targeted DEmRNA network and DElncRNA-DEmRNA co-expression network were constructed to further explore the roles of DElncRNAs in BA pathogenesis. The expression profiles of significant DEmRNAs were validated in Gene Expression Omnibus database. A total of 2,086 DEmRNAs and 184 DElncRNAs between BA and CC liver tissues were obtained. DEmRNAs were enriched in 521 Gene Ontology terms and 71 Kyoto Encyclopedia of Genes and Genomes terms which were mainly biological processes and metabolic pathways related to immune response and inflammatory response. A total of five hub proteins (TYRO protein tyrosine kinase binding protein, C-X-C motif chemokine ligand 8, pleckstrin, Toll-like receptor 8 and C-C motif chemokine receptor 5) were found in the PPI networks. A total of 31 DElncRNA-nearby-targeted DEmRNA pairs and 2,337 DElncRNA-DEmRNA co-expression pairs were obtained. The expression of DEmRNAs obtained from RNA sequencing were verified in GSE46960 dataset, generally. The present study identified key genes and lncRNAs participated in BA associated liver fibrosis, which may present a new avenue for understanding the patho-mechanism for hepatic fibrosis in BA.

Genetic Factors and Their Role in the Pathogenesis of Biliary Atresia.

Biliary Atresia, a common basis for neonatal cholestasis and primary indication for Liver Transplantation, accounts for 60% of pediatric Liver Transplantations. While the pathogenesis of Biliary Atresia remains obscure, abnormalities within bile ducts and the liver, inflammation, fibrosis and cilia defects are thought to comprise the pathological basis for this condition. The findings of genetic variants in Biliary Atresia, such as Copy Number Variations and Single Nucleotide Polymorphism, are considered as essential factors in the development of this condition. In this review, we summarize and analyze these Biliary Atresia variants from a perspective of their pathological characteristics. In conclusion, such analyses may offer novel insights into the pathogenesis of Biliary Atresia and provide a foundation for future studies directed toward a better understanding and treatment of Biliary Atresia.

Maternal risk factors associated with offspring biliary atresia: population-based study.

Biliary atresia (BA) is a progressive, idiopathic, fibro-obliterative disease of the intra and extrahepatic biliary tree. If untreated, it results in severe liver injury and death. The etiology and pathogenesis of BA remain unclear. Few studies have investigated the association between maternal illness/drug use and the occurrence of BA in offspring. We used the data from the Birth Certificate Application of Taiwan and linked to National Health Insurance Research Database and Taiwan Maternal and Child Health Database for the years 2004 to 2017 (N = 1,647,231) on 2022/03, and identified BA cases according to diagnosis and procedure code. A total of 285 BA cases were identified. Mothers with type 2 diabetes mellitus and non-dependent drug abuse had higher rates having BA children than non-BA children, with an odds ratio of 2.17 (95% confidence interval [CI] = 1.04-4.53) and OR: 3.02 (95% CI = 1.34-6.78), respectively. These results support the notion that BA occurrence is related to maternal reasons. Further studies should be designed to identify additional maternal and pregnancy risk factors and to understand the underlying pathophysiology. 1. The occurrence of offspring biliary atresia may be related to maternal illness/drug use. 2. Maternal drug abuse and type 2 diabetes mellitus pose a high risk for offspring biliary atresia. 3. If maternal etiology is found, biliary atresia might be a preventable disease.

Identification of Hub Genes and Immune Infiltration in Pediatric Biliary Atresia by Comprehensive Bioinformatics Analysis.

Background: Biliary atresia (BA) is the leading cause of pediatric liver failure and pediatric liver transplantation worldwide. Evidence suggests that the immune system plays a central role in the pathogenesis of BA. Methods: In this work, the novel immune-related genes between BA and normal samples were investigated based on weighted gene co-expression network analysis (WGCNA) and the deconvolution algorithm of CIBERSORT. Results: Specifically, 650 DEGs were identified between the BA and normal groups. The blue module was the most positively correlated with BA containing 3274 genes. Totally, 610 overlapping BA-related genes of DEGs and WGCNA were further used to identify IRGs. Three IRGs including VCAM1, HLA-DRA, and CD74 were finally identified as the candidate biomarkers. Particularly, the CD74 biomarker was discovered for the first as a potential immune biomarker for BA. Conclusions: Possibly, these 3 IRGs might serve as candidate biomarkers and guide the individualized treatment strategies for BA patients. Our results would provide great insights for a deeper understanding of both the occurrence and the treatment of BA.

Cellular senescence in the cholangiopathies: a driver of immunopathology and a novel therapeutic target.

The cholangiopathies are a group of liver diseases that affect cholangiocytes, the epithelial cells that line the bile ducts. Biliary atresia (BA), primary biliary cholangitis (PBC), and primary sclerosing cholangitis (PSC) are three cholangiopathies with significant immune-mediated pathogenesis where chronic inflammation and fibrosis lead to obliteration of bile ducts and eventual liver cirrhosis. Cellular senescence is a state of cell cycle arrest in which cells become resistant to apoptosis and profusely secrete a bioactive secretome. Recent evidence indicates that cholangiocyte senescence contributes to the pathogenesis of BA, PBC, and PSC. This review explores the role of cholangiocyte senescence in BA, PBC, and PSC, ascertains how cholangiocyte senescence may promote a senescence-associated immunopathology in these cholangiopathies, and provides the rationale for therapeutically targeting senescence as a treatment option for BA, PBC, and PSC.

A Comparative Study Between Cytomegalovirus Immunoglobulin M-Positive and CMV Immunoglobulin M-Negative Biliary Atresia in Infants Attending a Tertiary Care Hospital in Bangladesh.

PurposePerinatal cytomegalovirus (CMV) infection can lead to biliary atresia (BA) in different entities. This study aimed to compare the clinical, hematological, biochemical, and histological features of infants with BA based on their CMV immunoglobulin M (IgM) status at presentation.MethodsThis cross-sectional descriptive study was carried out between January 2019 and June 2020 at the Department of Pediatric Gastroenterology and Nutrition at the Bangabandhu Sheikh Mujib Medical University (BSMMU) in Dhaka. Forty-three patients with BA were selected purposively and categorized into either the CMV IgM-positive or CMV IgM-negative BA group. Categorical variables were compared using Fisher’s exact test and chi-square tests, while the Student’s t-test and Mann–Whitney U-test were used to compare continuous variables. For all statistical tests, a p-value <0.05 was considered statistically significant.ResultsThirty-three (76.7%) of the cases were between 2 and 3 months of age on admission. The clinical, hematological, and biochemical parameters did not differ significantly between the CMV IgM-positive and CMV IgM-negative BA groups. Most (50.0%) of the CMV IgM-positive cases had fibrosis stage F2, while 43.5% of the CMV IgM-negative cases had fibrosis stage F3, with no significant difference between the groups (p=0.391).ConclusionOur data shows no significant distinction between CMV IgM-positive and CMV IgM-negative BA, suggesting that CMV does not contribute to BA pathogenesis.

Altered T-Cell Receptor β-Chain and Lactate Dehydrogenase Are Associated With the Immune Pathogenesis of Biliary Atresia.

Background: Biliary atresia (BA) is considered to be an autoimmune-mediating inflammatory injury. The pathogenesis of BA has been proposed with the clonal transformation of T cells expressing analogous T-cell receptor β-chain variable regions (TRBVs).Methods: The TRBV profile of the peripheral blood mononuclear cells (PBMCs) in infants with BA and control infants (healthy donors, HDs), respectively, were characterized by using high-throughput sequencing (HTS). The diversity of T cells was analyzed based on the frequency of complementarity-determining region 3 (CDR3) or V(CDR3)J. Moreover, the correlation between absolute lymphocyte count (ALC) and lactate dehydrogenase (LDH) or diversity (clonality) indices, respectively, were analyzed for subjects with BA and HD.Results: The diversity indices of CDR3, V(CDR3)J in BA are lower than those in subjects with HD, in addition, there are significantly different levels of neutrophile, neutrophile/lymphocyte ratio (NLR), and LDH between groups of BA and HD. The correlation between ALC and diversity index is significant in subjects with HD but is not for subjects with BA. Conversely, the relationship between ALC and LDH is significant in subjects with BA but is not for subjects with HD. Moreover, 12 CDR3 motifs are deficient or lower expression in BA compared with that in the HD group.Conclusion: Our results demonstrate that the profile of TRBV repertoire is significantly different between subjects with BA and HD, and suggest that the immune imbalance and elevated LDH level are associated with the pathogenesis of BA. Moreover, the values of neutrophile, NLR, and LDH could be used for the differential diagnosis of BA.

The role of p.Val444Ala variant in the ABCB11 gene and susceptibility to biliary atresia in Vietnamese patients.

Biliary atresia (BA) is the most serious type of obstructive cholangiopathy that occurs in infants. BA can be the cause of death in children under 2 years if untreated early. However, the etiology of the disease is not known. BA is considered to be the result of the destruction of the bile duct system including the accumulation of bile acids. The bile salt export pump, a transporter protein encoded by the ABCB11 gene, plays the main role in the exportation and accumulation of bile acids. The p.Val444Ala variant in this gene is known to be associated with many cholestatic diseases. However, to date no study have been performed to evaluate the association of this variant with susceptibility to the risk of BA. In this study, we aimed to identify the frequency of p.Val444Ala variant and the risk of BA in Vietnamese patients.The polymerase chain reaction (PCR)– restriction fragment length polymorphism method was used to determine the frequency of alleles c.1331T>C (p.Val444Ala, rs2287622) in the ABCB11 gene in 266 Vietnamese patients with BA and 150 healthy people. The gene segment containing the variant was amplified by PCR with specific primers, after that the PCR products were cut by HaeIII restriction enzyme and analyzed on agarose gel to determine the genotypes. The frequency of alleles was assessed statistically to determine the association between these alleles and the risk of disease in patients.In our study, the frequency of alleles c.1331T>C (p.Val444Ala, rs2287622) in the ABCB11 gene was investigated the first time in the patients with BA. The results showed that CC and TC genotypes were significantly different between BA patients and healthy people (P < .01), and the C allele was associated with an increased risk of BA (odds ratio = 2.47; 95% confidence interval: 1.84–3.32; P < .01). The initial results of clinical, biochemical, and genetic analysis in our study suggested that the p.Val444Ala variant in the ABCB11 gene may be a susceptibility factor for the disease in Vietnamese patients with BA. These results provided new insights into the role of this ABCB11 variant in the pathogenesis of BA.

Identification of a wide spectrum of ciliary gene mutations in nonsyndromic biliary atresia patients implicates ciliary dysfunction as a novel disease mechanism.

BackgroundBiliary atresia (BA) is the most common obstructive cholangiopathy in neonates, often progressing to end-stage cirrhosis. BA pathogenesis is believed to be multifactorial, but the genetic contribution, especially for nonsyndromic BA (common form: > 85%) remains poorly defined.MethodsWe conducted whole exome sequencing on 89 nonsyndromic BA trios to identify rare variants contributing to BA etiology. Functional evaluation using patients’ liver biopsies, human cell and zebrafish models were performed. Clinical impact on respiratory system was assessed with clinical evaluation, nasal nitric oxide (nNO), high speed video analysis and transmission electron microscopy.FindingsWe detected rare, deleterious de novo or biallelic variants in liver-expressed ciliary genes in 31.5% (28/89) of the BA patients. Burden test revealed 2.6-fold (odds ratio (OR) [95% confidence intervals (CI)]= 2.58 [1.15–6.07], adjusted p = 0.034) over-representation of rare, deleterious mutations in liver-expressed ciliary gene set in patients compared to controls. Functional analyses further demonstrated absence of cilia in the BA livers with KIF3B and TTC17 mutations, and knockdown of PCNT, KIF3B and TTC17 in human control fibroblasts and cholangiocytes resulted in reduced number of cilia. Additionally, CRISPR/Cas9-engineered zebrafish knockouts of KIF3B, PCNT and TTC17 displayed reduced biliary flow. Abnormally low level of nNO was detected in 80% (8/10) of BA patients carrying deleterious ciliary mutations, implicating the intrinsic ciliary defects.InterpretationOur findings support strong genetic susceptibility for nonsyndromic BA. Ciliary gene mutations leading to cholangiocyte cilia malformation and dysfunction could be a key biological mechanism in BA pathogenesis.FundingThe study is supported by General Research Fund, HMRF Commissioned Paediatric Research at HKCH and Li Ka Shing Faculty of Medicine Enhanced New Staff Start-up Fund.

High Mobility Group Box 1 Release by Cholangiocytes Governs Biliary Atresia Pathogenesis and Correlates With Increases in Afflicted Infants.

Background and AimsBiliary atresia (BA) is a devastating cholangiopathy of infancy. Upon diagnosis, surgical reconstruction by Kasai hepatoportoenterostomy (HPE) restores biliary drainage in a subset of patients, but most patients develop fibrosis and progress to end‐stage liver disease requiring liver transplantation for survival. In the murine model of BA, rhesus rotavirus (RRV) infection of newborn pups results in a cholangiopathy paralleling that of human BA. High‐mobility group box 1 (HMGB1) is an important member of the danger‐associated molecular patterns capable of mediating inflammation during infection‐associated responses. In this study, we investigated the role of HMGB1 in BA pathogenesis.Approach and ResultsIn cholangiocytes, RRV induced the expression and release of HMGB1 through the p38 mitogen‐activated protein kinase signaling pathway, and inhibition of p38 blocked HMGB1 release. Treatment of cholangiocytes with ethyl pyruvate suppressed the release of HMGB1. Administration of glycyrrhizin in vivo decreased symptoms and increased survival in the murine model of BA. HMGB1 levels were measured in serum obtained from infants with BA enrolled in the PROBE and START studies conducted by the Childhood Liver Disease Research Network. High HMGB1 levels were found in a subset of patients at the time of HPE. These patients had higher bilirubin levels 3 months post‐HPE and a lower survival of their native liver at 2 years.ConclusionsThese results suggest that HMGB1 plays a role in virus induced BA pathogenesis and could be a target for therapeutic interventions in a subset of patients with BA and high HMGB1.

Necroptosis in biliary atresia of the liver.

Biliary atresia (BA) is characterized by the occlusion of extrahepatic bile ducts due to sclerosing inflammation. Necroptosis is a recently characterized form of programmed cell death but has not been examined in BA. We, therefore, explored the potential involvement of necroptosis in the pathogenesis of BA by evaluating the correlation between necroptosis-related factors and clinicopathological features of BA patients. We studied liver biopsy specimens of 59 patients with BA and 30 with congenital biliary dilatation (CBD). We also evaluated 14 surgical BA cases, who eventually underwent liver transplantation and 9 normal liver from neonates and infants obtained at autopsy. Necroptosis-related factors including toll-like receptor 3 (TLR3), receptor-interacting protein kinase1 (RIP1), receptor-interacting protein kinase3 (RIP3), mixed lineage kinase domain-like (MLKL), and phosphorylated mixed lineage kinase domain-like (pMLKL) in these liver specimens were immunolocalized. TLR3, RIP1, MLKL in the intrahepatic cholangiocytes was significantly higher in BA than CBD. pMLKL immunoreactivity was significantly greater at an earlier age of BA patients. The native liver survival period was significantly prolonged in the high RIP3 group. The low RIP3 status could serve as an adverse clinical prognostic factor for the native liver survival among the necroptosis-related factors examined in this study.

Diminution of the Primary Cilia From the Intrahepatic Cholangiocytes in a Pediatric Choledochal Cyst.

Choledochal cyst (CC) is a cystic disease predominantly involving the extrahepatic biliary tree. Biliary atresia (BA), another disorder of the extrahepatic biliary tree, is sometimes considered to be in the same spectrum as pediatric CC. Recently, the absence and/or the structural abnormality of the primary cilia in the intrahepatic cholangiocytes have been implicated in the pathogenesis of BA. We aimed to evaluate the expression of primary ciliary proteins in the intrahepatic cholangiocytes in cases of pediatric CC and compare it with normal control and BA. We performed immunohistochemistry for primary ciliary proteins (acetylated-α-tubulin and double-cortin domain containing 2) on the liver biopsies of control liver (n=5), pediatric CC (n=13), and BA (n=14). We also compared the expression with various clinical, biochemical, histopathologic (portal fibroinflammation and ductal plate malformation), and immunohistochemical (proliferative index) data. There was significant loss of primary cilia from the intrahepatic cholangiocytes in cases of CC and BA as compared with the normal control by both immunostains (CC: P=0.003 and 0.001, respectively; BA: P=0.001 and 0.001, respectively). There was no significant difference between the CC and BA in terms of ciliary protein loss. The loss of the ciliary proteins occurred irrespective of the proliferative (MIB-1 labeling) index, portal fibroinflammation, or ductal plate malformation. The loss of cilia did not correlate with the clinical follow-up in cases of pediatric CC. The loss of primary cilia from the intrahepatic cholangiocytes may be crucial in the etiopathogenesis of pediatric CC.

Biliary Atresia Subtypes Based on the Immune Cells and Genes Expressions by Unsupervised Clustering Imply the Underlying Pathogenesis

Background: The pathogenesis and phenotyping of biliary atresia (BA) is not clear. We aimed to provide a new view to identify the subtype of BA and explore the underlying pathogenesis. Methods: We collected the BA microarray datasets from Gene Expression Omnibus (GEO), and performed an unsupervised cluster analysis on abundance of immune cells and gene expressions and identified de novo classified BA patients. Then, we explored the possible molecular mechanisms of each subtype by functional enrichment analysis and the hub genes. Findings: First we classified BA patients into three subtypes and validated in GSE15235 dataset. Then we identified representative genes and immune cells for each subtype. The result of functional enrichment analysis and hub genes in subtype 2 was correlated with immune receptor activity, cytokine receptor, signaling by interleukin including IL-4, IL-13, IL-10, viral protein interaction, this may infer subtype 2 was corresponded to cytomegalovirus-associated variants of the clinical phenotyping classifies and modulation of interleukin signaling may represent potential novel therapeutic approaches. Neutrophil degranulation by Reactome pathway analysis existed in all the subtypes. Interpretation: The subtypes and function analysis identified in this study help us understand the underlying pathogenesis of BA, and provide new ideas for basic and clinical study. Funding: This work was supported by a grant from the National Key R&D Program of China (2018YFC1315400) and 5010 Project of Sun Yat-sen University (No.2018024). Declaration of Interest: None to declare.

Identification of a Wide Spectrum of Ciliary Gene Mutations in Nonsyndromic Biliary Atresia Patients Implicates Ciliary Dysfunction as a Novel Disease Mechanism

Background: Biliary atresia (BA) is the most common obstructive cholangiopathy in neonates, often progressing to end-stage cirrhosis. BA pathogenesis is believed to be multifactorial, but the genetic contribution, especially for nonsyndromic BA (common form:>85%) remains poorly defined. Methods: We conducted whole exome sequencing on 89 nonsyndromic BA trios to identify rare variants contributing to BA etiology. Functional evaluation using patients’ liver biopsies, human cell and zebrafish models were performed. Clinical impact on respiratory system was assessed with clinical evaluation, nasal nitric oxide (nNO), high speed video analysis and transmission electron microscopy. Findings: We detected rare, deleterious de novo or biallelic variants in liver-expressed ciliary genes in 31.5% (28/89) of the BA patients. Burden test revealed 2.6-fold (odds ratio (OR) [95% confidence intervals (CI)]=2.58 [1.15-6.07], adjusted p=0.034) over-representation of rare, deleterious mutations in liver-expressed ciliary geneset in patients compared to controls. Functional analyses further demonstrated absence of cilia in the BA livers with KIF3B and TTC17 mutations, and knockdown of PCNT, KIF3B and TTC17 in human control fibroblasts resulted in reduced number of cilia. Additionally, CRISPR/Cas9-engineered zebrafish knockouts of KIF3B, PCNT and TTC17 displayed reduced biliary flow. Abnormally low level of nNO was detected in 80% (8/10) of BA patients carrying deleterious ciliary mutations, implicating the intrinsic ciliary defects. Interpretation: Our findings support strong genetic susceptibility for nonsyndromic BA. Ciliary gene mutations leading to cholangiocyte cilia malformation and dysfunction could be a key biological mechanism in BA pathogenesis. Funding Statement: This work was supported by research grants from the Hong Kong General Research Fund (grant no. 17107314 and 766112 to M.M.G.B., 17105119 to V.C.H.L.) and Li Ka Shing Donation Account - Enhanced New Staff Start-up Packages to P.H.Y.C. Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: The study protocol was approved by the Institutional Review Board of the University of Hong Kong - Hospital Authority Hong Kong West Cluster (UW 05-282 T/945). Informed consent, or informed parental consent for those under 18 years old, was obtained from all participants.

A Higher Incidence of Isolated Biliary Atresia in Rural Areas: Results From an Epidemiological Study in The Netherlands.

Environmental factors may be involved in the pathogenesis of biliary atresia (BA). This epidemiological study aimed to analyze the relationships between the incidence of BA, the incidence of confirmed viral or bacterial infections and population density, and geographical and temporal clustering of BA in the Netherlands. Correlations between the monthly incidence of BA and the number of confirmed infections were assessed. BA incidence per province was calculated and compared to the province with highest population density. Birthplaces were classified as rural or urban. Temporal clustering of month of birth and month of conception were analyzed. We performed analyses for isolated BA (IBA) and syndromic BA (SBA) separately. Chi2, logistic regression, and Walter and Elwood test were used. A total of 262 IBA and 49 SBA patients, born between 1987 and 2018, were included. IBA incidence correlated to the number of confirmed infections of, for example, Chlamydia trachomatis (R = 0.14; P = 0.02) and adenovirus (R = 0.22; P = 0.005). We observed a higher incidence of IBA (0.75/10,000; odds ratio [OR] = 1.86; P = 0.04) and SBA (0.27/10,000; OR = 6.91; P = 0.001) in Groningen and a higher incidence of SBA in Gelderland (0.13/10,000; OR = 3.35; P = 0.03). IBA incidence was 68% higher in rural (0.67/10,000) versus urban areas (0.40/10,000) (P = 0.02). The estimated month of conception of patients with SBA clustered in November (85% increase compared to average SBA incidence [0.09/10,000; P = 0.04]). IBA incidence correlated weakly with national confirmed infections. IBA and SBA incidence varied geographically in the Netherlands. IBA incidence was higher in rural than in urban areas, which may be explained decreased exposure to pathogens. Our results provide support for a role of environmental factors in the pathogenesis of IBA.

YAP Activation and Implications in Patients and a Mouse Model of Biliary Atresia.

Background and Aim: Biliary atresia (BA), an inflammatory destruction of the bile ducts, leads to liver fibrosis in infants and accounts for half of cases undergoing pediatric liver transplantation. Yes-associated protein (YAP), an effector of the Hippo signaling pathway, is critical in maintaining identities of bile ductal cells. Here, we evaluated the expression of YAP and YAP target genes in BA livers and a rhesus rotavirus (RRV)-induced BA mice model.Methods: Liver specimens collected from 200 BA patients were compared with those of 30 non-BA patients. Model mice liver tissues were also collected. The expression of YAP and YAP target genes were measured by transfection, RNA-seq, immunohistochemistry, immunoblot, and quantitative PCR. Masson's trichrome staining and the Biliary Atresia Research Consortium (BARC) system were utilized to score liver fibrosis status.Results: The expression of YAP is elevated and positively correlated with fibrosis in BA livers. Moreover, ANKRD1, which is identified as the target gene of YAP, is also highly expressed in BA livers. Consistent with clinical data, YAP and ANKRD1 are significantly upregulated in RRV-induced BA mouse model.Conclusions: YAP expression is closely correlated with the bile duct hyperplasia and liver fibrosis, and may serve as an indicator for liver fibrosis and BA progression. This study indicates an involvement of the Hippo signaling pathway in the development of BA, and the YAP induced ANKRD1 expression may also be related to bile duct hyperplasia in BA. This provides a new direction for more in-depth exploration of the etiology and pathogenesis of biliary atresia.