Developing Biliary Atresia-like Model by Treating Human Liver Organoids with Polyinosinic:Polycytidylic Acid (Poly (I:C)).

Background: We explored the feasibility of creating BA-like organoids by treating human liver organoids with Polyinosinic:Polycytidylic acid (Poly I:C). Methods: Organoids were developed from the liver parenchyma collected during Kasai portoenterostomy (BA) and surgery for other liver disorders (non-BA). The non-BA organoids were co-cultured with poly I:C (40 µg/mL). The organoid morphology from both samples was compared on day 17. RNA-sequencing was performed to examine the transcriptomic differences. Results: Non-BA liver organoids developed into well-expanded spherical organoids with a single-cell layer of epithelial cells and a single vacuole inside. After poly I:C treatment, the majority of these organoids developed into an aberrant morphology with a high index of similarity to BA organoids which are multi-vacuoled and/or unexpanded. RNA-sequencing analysis revealed that 19 inflammatory genes were commonly expressed in both groups. Conditional cluster analysis revealed several genes (SOCS6, SOCS6.1, ARAF, CAMK2G, GNA1C, ITGA2, PRKACA, PTEN) that are involved in immune-mediated signaling pathway had a distinct pattern of expression in the poly I:C treated organoids. This resembled the expression pattern in BA organoids (p < 0.05). Conclusions: Poly I:C treated human liver organoids exhibit morphology and genetic signature highly compatible to organoids developed from BA liver samples. They are potential research materials to study immune-mediated inflammation in BA.

Current Understanding in the Clinical Characteristics and Molecular Mechanisms in Different Subtypes of Biliary Atresia.

Biliary atresia is a severe obliterative cholangiopathy in early infancy that is by far the most common cause of surgical jaundice and the most common indicator for liver transplantation in children. With the advanced knowledge gained from different clinical trials and the development of research models, a more precise clinical classification of BA (i.e., isolated BA (IBA), cystic BA (CBA), syndromic BA (SBA), and cytomegalovirus-associated BA (CMVBA)) is proposed. Different BA subtypes have similar yet distinguishable clinical manifestations. The clinical and etiological heterogeneity leads to dramatically different prognoses; hence, treatment needs to be specific. In this study, we reviewed the clinical characteristics of different BA subtypes and revealed the molecular mechanisms of their developmental contributors. We aimed to highlight the differences among these various subtypes of BA which ultimately contribute to the development of a specific management protocol for each subtype.

Beta-amyloid deposition around hepatic bile ducts is a novel pathobiological and diagnostic feature of biliary atresia.

BACKGROUND AND AIMS: Biliary atresia (BA) is a poorly understood and devastating obstructive bile duct disease of newborns. It is often diagnosed late, is incurable and frequently requires liver transplantation. In this study, we aimed to investigate the underlying pathogenesis and molecular signatures associated with BA. METHODS: We combined organoid and transcriptomic analysis to gain new insights into BA pathobiology using patient samples and a mouse model of BA. RESULTS: Liver organoids derived from patients with BA and a rhesus rotavirus A-infected mouse model of BA, exhibited aberrant morphology and disturbed apical-basal organization. Transcriptomic analysis of BA organoids revealed a shift from cholangiocyte to hepatocyte transcriptional signatures and altered beta-amyloid-related gene expression. Beta-amyloid accumulation was observed around the bile ducts in BA livers and exposure to beta-amyloid induced the aberrant morphology in control organoids. CONCLUSION: The novel observation that beta-amyloid accumulates around bile ducts in the livers of patients with BA has important pathobiological implications, as well as diagnostic potential. LAY SUMMARY: Biliary atresia is a poorly understood and devastating obstructive bile duct disease of newborns. It is often diagnosed late, is incurable and frequently requires liver transplantation. Using human and mouse 'liver mini-organs in the dish', we unexpectedly identified beta-amyloid deposition - the main pathological feature of Alzheimer's disease and cerebral amyloid angiopathy - around bile ducts in livers from patients with biliary atresia. This finding reveals a novel pathogenic mechanism that could have important diagnostic and therapeutic implications.

Genome-wide copy number variation study in anorectal malformations.

Anorectal malformations (ARMs, congenital obstruction of the anal opening) are among the most common birth defects requiring surgical treatment (2-5/10 000 live-births) and carry significant chronic morbidity. ARMs present either as isolated or as part of the phenotypic spectrum of some chromosomal abnormalities or monogenic syndromes. The etiology is unknown. To assess the genetic contribution to ARMs, we investigated single-nucleotide polymorphisms and copy number variations (CNVs) at genome-wide scale. A total of 363 Han Chinese sporadic ARM patients and 4006 Han Chinese controls were included. Overall, we detected a 1.3-fold significant excess of rare CNVs in patients. Stratification of patients by presence/absence of other congenital anomalies showed that while syndromic ARM patients carried significantly longer rare duplications than controls (P = 0.049), non-syndromic patients were enriched with both rare deletions and duplications when compared with controls (P = 0.00031). Twelve chromosomal aberrations and 114 rare CNVs were observed in patients but not in 868 controls nor 11 943 healthy individuals from the Database of Genomic Variants. Importantly, these aberrations were observed in isolated ARM patients. Gene-based analysis revealed 79 genes interfered by CNVs in patients only. In particular, we identified a de novo DKK4 duplication. DKK4 is a member of the WNT signaling pathway which is involved in the development of the anorectal region. In mice, Wnt disruption results in ARMs. Our data suggest a role for rare CNVs not only in syndromic but also in isolated ARM patients and provide a list of plausible candidate genes for the disorder.

Genome-wide copy number analysis uncovers a new HSCR gene: NRG3.

Hirschsprung disease (HSCR) is a congenital disorder characterized by aganglionosis of the distal intestine. To assess the contribution of copy number variants (CNVs) to HSCR, we analysed the data generated from our previous genome-wide association study on HSCR patients, whereby we identified NRG1 as a new HSCR susceptibility locus. Analysis of 129 Chinese patients and 331 ethnically matched controls showed that HSCR patients have a greater burden of rare CNVs (p = 1.50 × 10(-5)), particularly for those encompassing genes (p = 5.00 × 10(-6)). Our study identified 246 rare-genic CNVs exclusive to patients. Among those, we detected a NRG3 deletion (p = 1.64 × 10(-3)). Subsequent follow-up (96 additional patients and 220 controls) on NRG3 revealed 9 deletions (combined p = 3.36 × 10(-5)) and 2 de novo duplications among patients and two deletions among controls. Importantly, NRG3 is a paralog of NRG1. Stratification of patients by presence/absence of HSCR-associated syndromes showed that while syndromic-HSCR patients carried significantly longer CNVs than the non-syndromic or controls (p = 1.50 × 10(-5)), non-syndromic patients were enriched in CNV number when compared to controls (p = 4.00 × 10(-6)) or the syndromic counterpart. Our results suggest a role for NRG3 in HSCR etiology and provide insights into the relative contribution of structural variants in both syndromic and non-syndromic HSCR. This would be the first genome-wide catalog of copy number variants identified in HSCR.

Common genetic variants regulating ADD3 gene expression alter biliary atresia risk.

BACKGROUND & AIMS: Biliary atresia (BA) is a rare and most severe cholestatic disease in neonates, but the pathogenic mechanisms are unknown. Through a previous genome wide association study (GWAS) on Han Chinese, we discovered association of the 10q24.2 region encompassing ADD3 and XPNPEP1 genes, which was replicated in Chinese and Thai populations. This study aims to fully characterize the genetic architecture at 10q24.2 and to reveal the link between the genetic variants and BA. METHODS: We genotyped 107 single nucleotide polymorphisms (SNPs) in 10q24.2 in 339 Han Chinese patients and 401 matched controls using Sequenom. Exhaustive follow-up studies of the association signals were performed. RESULTS: The combined BA-association p-value of the GWAS SNP (rs17095355) achieved 6.06×10(-10). Further, we revealed the common risk haplotype encompassing 5 tagging-SNPs, capturing the risk-predisposing alleles in 10q24.2 [p=5.32×10(-11); odds ratio, OR: 2.38; confidence interval, CI: (2.14-2.62)]. Through Sanger sequencing, no deleterious rare variants (RVs) residing in the risk haplotype were found, dismissing the theory of "synthetic" association. Moreover, in bioinformatics and in vivo genotype-expression investigations, the BA-associated potentially regulatory SNPs correlated with ADD3 gene expression (n=36; p=0.0030). Remarkably, the risk haplotype frequency coincides with BA incidences in the population, and, positive selection (favoring the derived alleles that arose from mutations) was evident at the ADD3 locus, suggesting a possible role for the BA-associated common variants in shaping the general population diversity. CONCLUSIONS: Common genetic variants in 10q24.2 can alter BA risk by regulating ADD3 expression levels in the liver, and may exert an effect on disease epidemiology and on the general population.

Temporal and spatial development of intestinal smooth muscle layers of human embryos and fetuses.

The sequential occurrence of three layers of smooth muscle layers (SML) in human embryos and fetus is not known. Here, we investigated the process of gut SML development in human embryos and fetuses and compared the morphology of SML in fetuses and neonates. The H&E, Masson trichrome staining, and Immunohistochemistry were conducted on 6-12 gestation week human embryos and fetuses and on normal neonatal intestine. We showed that no lumen was seen in 6-7th gestation week embryonic gut, neither gut wall nor SML was developed in this period. In 8-9th gestation week embryonic and fetal gut, primitive inner circular SML (IC-SML) was identified in a narrow and discontinuous gut lumen with some vacuoles. In 10th gestation week fetal gut, the outer longitudinal SML (OL-SML) in gut wall was clearly identifiable, both the inner and outer SML expressed α-SMA. In 11-12th gestation week fetal gut, in addition to the IC-SML and OL-SML, the muscularis mucosae started to develop as revealed by α-SMA immune-reactivity beneath the developing mucosal epithelial layer. Comparing with the gut of fetuses of 11-12th week of gestation, the muscularis mucosae, IC-SML, and OL-SML of neonatal intestine displayed different morphology, including branching into glands of lamina propria in mucosa and increased thickness. In conclusions, in the human developing gut between week-8 to week-12 of gestation, the IC-SML develops and forms at week-8, followed by the formation of OL-SML at week-10, and the muscularis mucosae develops and forms last at week-12.

Does the Choice of Suture Materials Affect the Vas Deferens after Hernia Ring Closure in Inguinal Hernia Surgery: An Experimental Study in Rats.

INTRODUCTION: Laparoscopic inguinal hernia repair is a commonly performed procedure in children. Currently, monofilament polypropylene and braided silk are the two most frequently used materials. Studies have suggested more tissue inflammatory reactions with the use of multifilament non-absorbable sutures. However, little is known about the effects of suture materials on adjacent vas deferens. The aim of this experiment was to compare the effect of non-absorbable monofilament and multifilament sutures on vas deferens in laparoscopic hernia repair. METHODS: All animal operations were performed by a single surgeon under aseptic conditions and anaesthesia. Ten male Sprague Dawley rats were divided into two groups. In Group I, "hernia repair" was performed using 5.0 Silk. In Group II, polypropylene sutures (Prolene®; Ethicon, Somerville, N.J., USA) were used. All animals also received sham operations in the left groin as a control. After 14 days, the animals were euthanised and a segment of vas deferens just adjacent to the suture was excised for histological review by an experienced pathologist who was blind to the treatment groups of the respective specimens. RESULTS: The body sizes of the rats in each group were comparable. Group I had significantly smaller vas deferens than Group II (diameter: 0.2 vs. 0.6 ± 0.2, p = 0.005). Silk sutures appeared to cause more tissue adhesion than Prolene® sutures, as graded by blind assessors (adhesion grade: 2.8 ± 1.3 vs. 1.8 ± 0.8, p = 0.1), although this did not reach statistical significance. There was no significant difference in the histological fibrosis score and inflammation score. CONCLUSION: The only effect of non-absorbable sutures on vas deferens in this rat model was the reduced cross-sectional area of vas deferens and increased tissue adhesion when using silk sutures. However, there was no significant histological difference in inflammation or fibrosis caused by either material.

Identification of cancer-related genes FGFR2 and CEBPB in choledochal cyst via RNA sequencing of patient-derived liver organoids.

BACKGROUND: Choledochal cysts (CC) are congenital bile duct anomalies with 6-30% risk for developing bile duct cancer. However, the molecular mechanisms underlying cancer risk of CC are unknown. We sought to identify the gene expression changes underlying the cancer risk of CC patients. METHODS: Liver organoids (n = 51) were generated from liver/bile duct biopsies of CC (n = 7; type I) and hepatoblastoma (n = 5; HB: non-tumor & tumor) for RNA sequencing. Bioinformatics analysis was conducted to identify differentially expressed cancer-related genes in CC and controls. We compared CC with non-cancerous and cancerous controls, normal adjacent non-tumor region of hepatoblastoma (HB) liver as non-cancerous control and tumor region as non-CC cancer control (HB-tumor). Reverse transcription real-time quantitative PCR (RT-qPCR) verification and immunohistochemistry of selected genes was conducted in additional CC and HB liver biopsies. FINDINGS: HB non-tumor and HB tumor organoids displayed distinct gene expression profiles. Expression profiling separated CC organoids into two clusters, one overlapping with HB non-tumor and the other one with HB tumor organoids. Genes selected based on their log2FoldChange values for RT-qPCR verification in 31 CC and 11 HB non-tumor liver tissues revealed significantly elevated expression of FGFR2 in 7 and CEBPB in 2 CC liver tissues (CC vs HB: 4.082 vs. 0.7671, p<0.01; 2.506 vs. 1.210, p<0.01). Distinctive positive staining in bile ducts were seen in CC, HB tumor and non-tumor liver tissues for FGFR2 and CEBPB. Percentages of CEBPB-immuno-positive or FGFR2-immuno-positive bile duct cells in CC and HB-tumor liver were higher than that in HB non-tumor liver. INTERPRETATION: The study identified dysregulated genes related to cancer pathways in CC patients suggesting cancer risk. The findings suggest that the elevated expression of FGFR2 and CEBPB in liver may contribute to cancer development in CC patients.

Therapeutic potential of in utero mesenchymal stem cell (MSCs) transplantation in rat foetuses with spina bifida aperta.

Neural tube defects (NTDs) are complex congenital malformations resulting from incomplete neurulation in embryo. Despite surgical repair of the defect, most of the patients who survive with NTDs have a multiple system handicap due to neuron deficiency of the defective spinal cord. In this study, we successfully devised a prenatal surgical approach and transplanted mesenchymal stem cells (MSCs) to foetal rat spinal column to treat retinoic acid induced NTDs in rat. Transplanted MSCs survived, grew and expressed markers of neurons, glia and myoblasts in the defective spinal cord. MSCs expressed and perhaps induced the surrounding spinal tissue to express neurotrophic factors. In addition, MSC reduced spinal tissue apoptosis in NTD. Our results suggested that prenatal MSC transplantation could treat spinal neuron deficiency in NTDs by the regeneration of neurons and reduced spinal neuron death in the defective spinal cord.

Reduced RET expression in gut tissue of individuals carrying risk alleles of Hirschsprung's disease.

Receptor tyrosine kinase (RET) single nucleotide polymorphisms (SNPs) are associated with the Hirschsprung's disease (HSCR). We investigated whether the amount of RET expressed in the ganglionic gut of human was dependent on the genotype of three regulatory SNPs (-5G>A rs10900296 and -1A>C rs10900297 in the promoter, and C>T rs2435357 in intron 1). We examined the effects of three regulatory SNPs on the RET gene expression in 67 human ganglionic gut tissues using quantitative real-time PCR. Also, 315 Chinese HSCR patients and 325 ethnically matched controls were genotyped for the three SNPs by polymerase chain reaction (PCR) and direct sequencing. The expression of RET mRNA in human gut tissue did indeed correlate with the genotypes of the individuals. The lowest RET expression was found for those individuals homozygous for the three risk alleles (A-C-T/A-C-T), and the highest for those homozygous for the 'wild-type' counterpart (G-A-C/G-A-C), with expression values ranging from 218.32 +/- 125.69 (mean +/- SE) in tissues from individuals carrying G-A-C/G-A-C to 31.42 +/- 8.42 for individuals carrying A-C-T/A-C-T (P = 0.018). As expected, alleles -5A, -1C and intron 1 T were associated with HSCR (P = 5.94 x 10(-31), 3.12 x 10(-24) and 5.94 x 10(-37), respectively) as was the haplotype encompassing the three associated alleles (A-C-T) when compared with the wild-type counterpart G-A-C (chi2 = 155.29, P << 0.0001). To our knowledge, this is the first RET expression genotype-phenotype correlation study conducted on human subjects to indicate common genetic variants in the regulatory region of RET may play a role in mediating susceptibility to HSCR, by conferring a significant reduction of the RET expression.

Genome-wide association study identifies a susceptibility locus for biliary atresia on 10q24.2.

Biliary atresia (BA) is characterized by the progressive fibrosclerosing obliteration of the extrahepatic biliary system during the first few weeks of life. Despite early diagnosis and prompt surgical intervention, the disease progresses to cirrhosis in many patients. The current theory for the pathogenesis of BA proposes that during the perinatal period, a still unknown exogenous factor meets the innate immune system of a genetically predisposed individual and induces an uncontrollable and potentially self-limiting immune response, which becomes manifest in liver fibrosis and atresia of the extrahepatic bile ducts. Genetic factors that could account for the disease, let alone for its high incidence in Chinese, are to be investigated. To identify BA susceptibility loci, we carried out a genome-wide association study (GWAS) using the Affymetrix 5.0 and 500 K marker sets. We genotyped nearly 500 000 single-nucleotide polymorphisms (SNPs) in 200 Chinese BA patients and 481 ethnically matched control subjects. The 10 most BA-associated SNPs from the GWAS were genotyped in an independent set of 124 BA and 90 control subjects. The strongest overall association was found for rs17095355 on 10q24, downstream XPNPEP1, a gene involved in the metabolism of inflammatory mediators. Allelic chi-square test P-value for the meta-analysis of the GWAS and replication results was 6.94 x 10(-9). The identification of putative BA susceptibility loci not only opens new fields of investigation into the mechanisms underlying BA but may also provide new clues for the development of preventive and curative strategies.

Genome-wide association study identifies NRG1 as a susceptibility locus for Hirschsprung's disease.

Hirschsprung's disease (HSCR), or aganglionic megacolon, is a congenital disorder characterized by the absence of enteric ganglia in variable portions of the distal intestine. RET is a well-established susceptibility locus, although existing evidence strongly suggests additional loci contributing to sporadic HSCR. To identify these additional genetic loci, we carried out a genome-wide association study using the Affymetrix 500K marker set. We successfully genotyped 293,836 SNPs in 181 Chinese subjects with sporadic HSCR and 346 ethnically matched control subjects. The SNPs most associated with HSCR were genotyped in an independent set of 190 HSCR and 510 control subjects. Aside from SNPs in RET, the strongest overall associations in plausible candidate genes were found for 2 SNPs located in intron 1 of the neuregulin1 gene (NRG1) on 8p12, with rs16879552 and rs7835688 yielding odds ratios of 1.68 [CI(95%):(1.40, 2.00), P = 1.80 x 10(-8)] and 1.98 [CI(95%):(1.59, 2.47), P = 1.12 x 10(-9)], respectively, for the heterozygous risk genotypes under an additive model. There was also a significant interaction between RET and NRG1 (P = 0.0095), increasing the odds ratio 2.3-fold to 19.53 for the RET rs2435357 risk genotype (TT) in the presence of the NRG1 rs7835688 heterozygote, indicating that NRG1 is a modifier of HSRC penetrance. Our highly significant association findings are backed-up by the important role of NRG1 as regulator of the development of the enteric ganglia precursors. The identification of NRG1 as an additional HSCR susceptibility locus not only opens unique fields of investigation into the mechanisms underlying the HSCR pathology, but also the mechanisms by which a discrete number of loci interact with each other to cause disease.

Pathogenesis of Choledochal Cyst: Insights from Genomics and Transcriptomics.

Choledochal cysts (CC) is characterized by extra- and/or intra-hepatic b\ile duct dilations. There are two main theories, "pancreaticobiliary maljunction" and "congenital stenosis of bile ducts" proposed for the pathogenesis of CC. Although family cases or CC associated with other anomalies have been reported, the molecular pathogenesis of CC is still poorly understood. Recent advances in transcriptomics and genomics analysis platforms have unveiled key expression signatures/genes/signaling pathways in the pathogenesis of human diseases including CC. This review summarizes insights from genomics and transcriptomics studies into the pathogenesis of CC, with the aim to improve (i) our understanding of its underlying complex pathomechanisms, and (ii) clinical management of different subtypes of CC, in particular their associated hepatic fibrotic change and their risk of malignancy transformation.

Bioengineering Liver Organoids for Diseases Modelling and Transplantation.

Organoids as three-dimension (3D) cellular organizations partially mimic the physiological functions and micro-architecture of native tissues and organs, holding great potential for clinical applications. Advances in the identification of essential factors including physical cues and biochemical signals for controlling organoid development have contributed to the success of growing liver organoids from liver tissue and stem/progenitor cells. However, to recapitulate the physiological properties and the architecture of a native liver, one has to generate liver organoids that contain all the major liver cell types in correct proportions and relative 3D locations as found in a native liver. Recent advances in stem-cell-, biomaterial- and engineering-based approaches have been incorporated into conventional organoid culture methods to facilitate the development of a more sophisticated liver organoid culture resembling a near to native mini-liver in a dish. However, a comprehensive review on the recent advancement in the bioengineering liver organoid is still lacking. Here, we review the current liver organoid systems, focusing on the construction of the liver organoid system with various cell sources, the roles of growth factors for engineering liver organoids, as well as the recent advances in the bioengineering liver organoid disease models and their biomedical applications.

Gut lumen formation defect can cause intestinal atresia: evidence from histological studies of human embryos and intestinal atresia septum.

Intestinal atresia (IA), a common cause of neonatal intestinal obstruction, is a developmental defect, which disrupts the luminal continuity of the intestine. Here, we investigated (i) the process of lumen formation in human embryos; and (ii) how a defective lumen formation led to IA. We performed histological and histochemical study on 6-10 gestation week human embryos and on IA septal regions. To investigate the topology of embryonic intestine development, we conducted 3D reconstruction. We showed that a 6-7th gestation week embryonic gut has no lumen, but filled with mesenchyme cells and vacuoles of a monolayer of epithelial cells. A narrow gut lumen was formed by gestation week-9, the gut was filled with numerous vacuoles of different sizes, some vacuoles were merging with the developing embryonic gut wall. At gestation week-10, a prominent lumen was developed, only few vacuoles were present and were merging with the intestine wall. At IA septal regions, vacuoles were located in the submucous layer, covered by a single layer of epithelium without glandular structure, and surrounded with fibrous tissue. The mucosal epithelium was developed with lamina propria and basement membrane, but the submucosa and the longitudinal smooth muscle layers were not properly developed. Hence, the vacuoles in IA septum could represent a remnant of vacuoles of embryonic gut. In conclusion, the fusion of vacuoles with the developing intestine wall associates with the disappearance of vacuoles and gut lumen formation in human embryos, and perturbation of these developmental events could lead to IA.

Human liver organoid derived intra-hepatic bile duct cells support SARS-CoV-2 infection and replication.

Although the main route of infection for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the respiratory tract, liver injury is also commonly seen in many patients, as evidenced by deranged parenchymal liver enzymes. Furthermore, the severity of liver damage has been shown to correlate with higher mortality. Overall, the mechanism behind the liver injury remains unclear. We showed in this study that intra-hepatic bile duct cells could be grown using a human liver organoid platform. The cholangiocytes were not only susceptible to SARS-CoV-2 infection, they also supported efficient viral replication. We also showed that SARS-CoV-2 replication was much higher than SARS-CoV. Our findings suggested direct cytopathic viral damage being a mechanism for SARS-CoV-2 liver injury.

Plasma amyloid-beta levels correlated with impaired hepatic functions: An adjuvant biomarker for the diagnosis of biliary atresia.

Background: Biliary atresia (BA) is an infantile fibro-obstructive cholestatic disease with poor prognosis. An early diagnosis and timely Kasai portoenterostomy (KPE) improve clinical outcomes. Aggregation of amyloid-beta (Aß) around hepatic bile ducts has been discovered as a factor for BA pathogenesis, yet whether plasma Aß levels correlate with hepatic dysfunctions and could be a biomarker for BA remains unknown. Method: Plasma samples of 11 BA and 24 controls were collected for liver function test, Aß40 and Aß42 measurement by enzyme-linked immunosorbent assay (ELISA). Pearson's chi-squared test or Mann-Whitney U test was performed to assess differences between groups. Correlation between Aß42/Aß40 and liver function parameters was performed using Pearson analysis. The area under the receiver-operative characteristic (ROC) curve (area under curve; AUC) was measured to evaluate the diagnostic power of Aß42/Aß40 for BA. Diagnostic enhancement was further evaluated by binary regression ROC analysis of Aß42/Aß40 combined with other hepatic function parameters. Results: Plasma Aß42/Aß40 was elevated in BA patients. Aß42 displayed a weak positive correlation with γ-glutamyl transpeptidase (GGT) (Pearson's correlation = 0.349), while there was no correlation for Aß40 with hepatic functions. Aß42/Aß40 was moderately correlated with GGT, total bile acid (TBA), direct bilirubin (DBIL) (Pearson's correlation = 0.533, 0.475, 0.480), and weakly correlated with total bilirubin (TBIL) (Pearson's correlation = 0.337). Aß42/Aß40 showed an acceptable predictive power for cholestasis [AUC = 0.746 (95% CI: 0.552-0.941), p < 0.05]. Diagnostic powers of Aß42/Aß40 together with hepatic function parameters for cholestasis were markedly improved compared to any indicator alone. Neither Aß42/Aß40 nor hepatic function parameters displayed sufficient power in discriminating BA from choledochal cysts (CC); however, combinations of Aß42/Aß40 + GGT along with any other hepatic function parameters could differentiate BA from CC-cholestasis (AUC = 1.000, p < 0.05) with a cut-off value as 0.02371, -0.28387, -0.34583, 0.06224, 0.01040, 0.06808, and 0.05898, respectively. Conclusion: Aß42/Aß40 is a good indicator for cholestasis, but alone is insufficient for a distinction of BA from non-BA. However, Aß42/Aß40 combined with GGT and one other hepatic function parameter displayed a high predictive power as a screening test for jaundiced neonates who are more likely to be BA, enabling them to early intraoperative cholangiography for BA confirmation and KPE to improve surgical outcomes. However, a multi-centers validation is needed before introduction into daily clinical practice.

Biomarkers for the diagnosis and post-Kasai portoenterostomy prognosis of biliary atresia: a systematic review and meta-analysis.

To evaluate the accuracy of biomarkers for the early diagnosis of biliary atresia (BA) and prognostic stratification after Kasai portoenterostomy (KPE). We conducted a systematic review of PubMed, Web of Science, Embase, Scopus and OVID for English literature reporting BA biomarkers published before August 2020. Screening, data extraction, and quality assessment were performed in duplicate. A total of 51 eligible studies were included in the systematic review, and data from 12 (4182 subjects) were extracted for meta-analysis regarding the following 2 domains: (1) serum matrix metallopeptidase-7 (MMP-7), interleukin33 (IL-33) and γ-glutamyl transferase (GGT) to differentiate BA from non-BA; (2) the aspartate aminotransferase to platelet ratio index (APRi) to predict post-KPE liver fibrosis/cirrhosis. The summary sensitivity, specificity and area under the curve (AUC) of MMP-7 for diagnosing BA were 96%, 91% and 0.9847, respectively, and those of GGT were 80%, 79% and 0.9645, respectively. The summary sensitivity and specificity of IL-33 for diagnosing BA were 77% and 85%, respectively. The summary sensitivity and specificity of APRi for predicting post-KPE liver fibrosis were 61% and 80%, respectively, and the summary sensitivity, specificity and AUC of APRi for predicting post-KPE cirrhosis were 78%, 83% and 0.8729, respectively. Moreover, good evidence was shown in investigations of serum IL-18 and IL-33 in distinguishing BA from healthy controls, serum IL-18 for prognosis of post-KPE persistent jaundice, and serum hyaluronic acid and MMP-7 for prognosis of post-KPE significant liver fibrosis. MMP-7, IL-33 and GGT are useful biomarkers to assist in the diagnosis of BA. APRi might be used to predict post-KPE significant liver fibrosis and cirrhosis. These noninvasive biomarkers can be integrated into the management protocol of BA.

Deletion of interleukin enhancer binding factor 2 (ILF2) resulted in defective biliary development and bile flow blockage.

PURPOSE: Biliary atresia (BA) is a devastating obstructive bile duct disease of newborns. BA has the highest incidence in Asians (1/5000), and its pathogenesis is unclear. We identified BA-private rare copy number variants (CNVs; 22 duplications and 6 deletions). ILF2 gene locates in the chromosome region (Chr1:153410347-153,634,058) which was deleted in a nonsyndromic BA patient. However, it is still not known whether ILF2 plays a role in hepatobiliary development and its deletion impacts on the bile duct development. METHODS: To investigate if ILF2 is required for biliary development, we knock-out the zebrafish homologs of ILF2 by CRISPR/Cas9 approach, and discover that deletion of ILF2 causes a defective biliary development and a lack of bile flow from the liver to the gall bladder in zebrafish, which is a resemblance of phenotypes of BA. RESULTS: Our data indicate that ILF2 gene is required for biliary development; deletion of ILF2 impairs bile duct development and could contribute to BA pathogenesis. This will be the first study to functionally evaluate the genes interfered by BA-private CNVs in hepatobiliary development and in BA pathogenesis. CONCLUSIONS: Such functional study may reveal the potential value of these BA-private CNVs in the disease pathogenesis for BA. LEVEL OF EVIDENCE: N/A (animal and laboratory study).