Comprehensive Association Analyses of Extraintestinal Manifestations in Inflammatory Bowel Disease.

BACKGROUND & AIMS: Patients with inflammatory bowel disease (IBD) frequently develop extraintestinal manifestations (EIMs) that contribute substantially to morbidity. We assembled the largest multicohort data set to date to investigate the clinical, serologic, and genetic factors associated with EIM complications in IBD. METHODS: Data were available in 12,083 unrelated European ancestry IBD cases with presence or absence of EIMs (eg, ankylosing spondylitis [ankylosing spondylitis and sacroiliitis], primary sclerosing cholangitis [PSC], peripheral arthritis, and skin and ocular manifestations) across 4 cohorts (Cedars-Sinai Medical Center, National Institute for Diabetes and Digestive and Kidney Diseases IBD Genetics Consortium, Sinai Helmsley Alliance for Research Excellence Consortium, and Risk Stratification and Identification of Immunogenetic and Microbial Markers of Rapid Disease Progression in Children with Crohn's Disease cohort). Clinical and serologic parameters were analyzed by means of univariable and multivariable regression analyses using a mixed-effects model. Within-case logistic regression was performed to assess genetic associations. RESULTS: Most EIMs occurred more commonly in female subjects (overall EIM: P = 9.0E-05, odds ratio [OR], 1.2; 95% CI, 1.1-1.4), with CD (especially colonic disease location; P = 9.8E-09, OR, 1.7; 95% CI, 1.4-2.0), and in subjects who required surgery (both CD and UC; P = 3.6E-19, OR, 1.7; 95% CI, 1.5-1.9). Smoking increased risk of EIMs except for PSC, where there was a "protective" effect. Multiple serologic associations were observed, including with PSC (anti-nuclear cytoplasmic antibody; IgG and IgA, anti-Saccharomyces cerevisiae antibodies; and anti-flagellin) and any EIM (anti-nuclear cytoplasmic antibody; IgG and IgA, anti-Saccharomyces cerevisiae antibodies; and anti-Pseudomonas fluorescens-associated sequence). We identified genome-wide significant associations within major histocompatibility complex (ankylosing spondylitis and sacroiliitis, P = 1.4E-15; OR, 2.5; 95% CI, 2.0-3.1; PSC, P = 2.7E-10; OR, 2.8; 95% CI, 2.0-3.8; ocular, P = 2E-08, OR, 3.6; 95% CI, 2.3-5.6; and overall EIM, P = 8.4E-09; OR, 2.2; 95% CI, 1.7-2.9) and CPEB4 (skin, P = 2.7E-08; OR, 1.5; 95% CI, 1.3-1.8). Genetic associations implicated tumor necrosis factor, JAK-STAT, and IL6 as potential targets for EIMs. Contrary to previous reports, only 2% of our subjects had multiple EIMs and most co-occurrences were negatively correlated. CONCLUSIONS: We have identified demographic, clinical, and genetic associations with EIMs that revealed underlying mechanisms and implicated novel and existing drug targets-important steps toward a more personalized approach to IBD management.

HLA-DPA1*02:01~B1*01:01 is a risk haplotype for primary sclerosing cholangitis mediating activation of NKp44+ NK cells.

OBJECTIVE: Primary sclerosing cholangitis (PSC) is characterised by bile duct strictures and progressive liver disease, eventually requiring liver transplantation. Although the pathogenesis of PSC remains incompletely understood, strong associations with HLA-class II haplotypes have been described. As specific HLA-DP molecules can bind the activating NK-cell receptor NKp44, we investigated the role of HLA-DP/NKp44-interactions in PSC. DESIGN: Liver tissue, intrahepatic and peripheral blood lymphocytes of individuals with PSC and control individuals were characterised using flow cytometry, immunohistochemical and immunofluorescence analyses. HLA-DPA1 and HLA-DPB1 imputation and association analyses were performed in 3408 individuals with PSC and 34 213 controls. NK cell activation on NKp44/HLA-DP interactions was assessed in vitro using plate-bound HLA-DP molecules and HLA-DPB wildtype versus knock-out human cholangiocyte organoids. RESULTS: NKp44+NK cells were enriched in livers, and intrahepatic bile ducts of individuals with PSC showed higher expression of HLA-DP. HLA-DP haplotype analysis revealed a highly elevated PSC risk for HLA-DPA1*02:01~B1*01:01 (OR 1.99, p=6.7×10-50). Primary NKp44+NK cells exhibited significantly higher degranulation in response to plate-bound HLA-DPA1*02:01-DPB1*01:01 compared with control HLA-DP molecules, which were inhibited by anti-NKp44-blocking. Human cholangiocyte organoids expressing HLA-DPA1*02:01-DPB1*01:01 after IFN-γ-exposure demonstrated significantly increased binding to NKp44-Fc constructs compared with unstimulated controls. Importantly, HLA-DPA1*02:01-DPB1*01:01-expressing organoids increased degranulation of NKp44+NK cells compared with HLA-DPB1-KO organoids. CONCLUSION: Our studies identify a novel PSC risk haplotype HLA-DP A1*02:01~DPB1*01:01 and provide clinical and functional data implicating NKp44+NK cells that recognise HLA-DPA1*02:01-DPB1*01:01 expressed on cholangiocytes in PSC pathogenesis.

Sex-Dependent Differences in Cholestasis: Why Estrogen Signaling May Be a Key Pathophysiological Driver.

Primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) are cholestatic liver diseases that have significant clinical impact with debilitating symptoms and mortality. While PBC is predominantly seen in perimenopausal and postmenopausal women, men who are diagnosed with PBC have worse clinical outcomes and all-cause mortality. In contrast, 60% to 70% of patients with PSC are men; the data indicate that female sex may be an independent factor against PSC-related complications. These findings suggest a sex-dependent biological basis for these differences. Estrogen has been implicated in the pathogenesis of intrahepatic cholestasis of pregnancy and may induce cholestasis through a variety of interactions. However, it is unclear why some sexual dimorphic features may provide a protective effect despite known estrogen models that induce cholestasis. This article provides a brief introductory background and discusses the sexual dimorphism in clinical presentation in PSC and PBC. It also explores the role of estrogen signaling in pathogenesis and how it relates to intrahepatic cholestasis of pregnancy. Studies have already targeted certain molecules involved in estrogen signaling, and this review discusses these studies that identify estrogen-related receptor, estrogen receptor-α, estrogen receptor-ß, farnesoid X receptor, and mast cells as possible targets, in addition to long noncoding RNA H19-induced cholestasis and sexual dimorphism. It also explores these interactions and their role in the pathogenesis of PBC and PSC.

Identifying the genetic association between systemic lupus erythematosus and the risk of autoimmune liver diseases.

BACKGROUND: Previous studies on the relationship between systemic lupus erythematosus (SLE) and autoimmune liver diseases (AILDs) are inconclusive. Therefore, we employed Mendelian randomization (MR) to explore the causal associations between SLE and AILDs. METHODS: A two-sample MR analysis was performed using summary-level statistics sourced from genome-wide association study (GWAS) datasets. Inverse-variance weighting (IVW), MR‒Egger, and weighted median (WM) were further supported by several sensitivity analyses. RESULTS: We detected causal genetic associations between SLE and primary biliary cholangitis (PBC) (odds ratio (OR) = 1.31, 95% CI = 1.15-1.51, P < 0.01; adjusted OR = 1.63, 95% CI = 1.39-1.90, P < 0.01) and between SLE and primary sclerosing cholangitis (PSC) (OR = 1.09, 95% CI = 1.01-1.08, P = 0.03; adjusted OR = 1.10, 95% CI = 1.00-1.21, P = 0.04). No causal association was found between SLE and autoimmune hepatitis. CONCLUSIONS: We are the first to use MR analysis to explore the causal relationships between SLE and various AILDs, revealing an increased risk of PBC and PSC in individuals with SLE.

Prolonged administration of a secretin receptor antagonist inhibits biliary senescence and liver fibrosis in Mdr2 -/- mice.

BACKGROUND AND AIMS: Secretin (SCT) and secretin receptor (SR, only expressed on cholangiocytes within the liver) play key roles in modulating liver phenotypes. Forkhead box A2 (FoxA2) is required for normal bile duct homeostasis by preventing the excess of cholangiocyte proliferation. Short-term administration of the SR antagonist (SCT 5-27) decreased ductular reaction and liver fibrosis in bile duct ligated and Mdr2 -/- [primary sclerosing cholangitis (PSC), model] mice. We aimed to evaluate the effectiveness and risks of long-term SCT 5-27 treatment in Mdr2 -/- mice. APPROACH AND RESULTS: In vivo studies were performed in male wild-type and Mdr2 -/- mice treated with saline or SCT 5-27 for 3 months and human samples from late-stage PSC patients and healthy controls. Compared with controls, biliary SCT/SR expression and SCT serum levels increased in Mdr2 -/- mice and late-stage PSC patients. There was a significant increase in ductular reaction, biliary senescence, liver inflammation, angiogenesis, fibrosis, biliary expression of TGF-ß1/VEGF-A axis, and biliary phosphorylation of protein kinase A and ERK1/2 in Mdr2 -/- mice. The biliary expression of miR-125b and FoxA2 decreased in Mdr2 -/- compared with wild-type mice, which was reversed by long-term SCT 5-27 treatment. In vitro , SCT 5-27 treatment of a human biliary PSC cell line decreased proliferation and senescence and SR/TGF-ß1/VEGF-A axis but increased the expression of miR-125b and FoxA2. Downregulation of FoxA2 prevented SCT 5-27-induced reduction in biliary damage, whereas overexpression of FoxA2 reduced proliferation and senescence in the human PSC cell line. CONCLUSIONS: Modulating the SCT/SR axis may be critical for managing PSC.

CLDN1 Arg81His founder variant causes ichthyosis, leukocyte vacuoles, alopecia, and sclerosing cholangitis (ILVASC) syndrome in Moroccan Jews.

Neonatal ichthyosis and sclerosing cholangitis syndrome (NISCH), also known as ichthyosis, leukocyte vacuoles, alopecia, and sclerosing cholangitis (ILVASC), is an extremely rare disease of autosomal recessive inheritance, resulting from loss of function of the tight junction protein claudin-1. Its clinical presentation is highly variable, and is characterized by liver and ectodermal involvement. Although most ILVASC cases described to date were attributed to homozygous truncating variants in CLDN1, a single missense variant CLDN1 p.Arg81His, associated with isolated skin ichthyosis phenotype, has been recently reported in a family of Moroccan Jewish descent. We now describe seven patients with ILVASC, originating from four non consanguineous families of North African Jewish ancestry (including one previously reported family), harboring CLDN1 p.Arg81His variant, and broaden the phenotypic spectrum attributed to this variant to include teeth, hair, and liver/bile duct involvement, characteristic of ILVASC. Furthermore, we provide additional evidence for pathogenicity of the CLDN1 p.Arg81His variant by transmission electron microscopy of the affected skin, revealing distorted tight junction architecture, and show through haplotype analysis in the vicinity of the CLDN1 gene, that this variant represents a founder variant in Jews of Moroccan descent with an estimated carrier frequency of 1:220.

ANXA1 is identified as a key gene associated with high risk and T cell infiltration in primary sclerosing cholangitis.

BACKGROUND: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease, with unclear pathogenesis. Although immune disorders, especially T cell infiltration, are thought to play a vital role in PSC, the specific pathogenesis mechanisms remain incompletely understood. This study evaluated the potential key gene associated with the PSC pathogenesis and analyzed the associations of the key gene with prognosis and immune cell infiltration by combining bioinformatics analysis and experimental verification. METHODS: Transcriptome data of PSC and normal human liver tissues (GSE159676) were obtained from the gene expression omnibus database. Differentially expressed genes (DEGs) were identified, and differences in biological states were analyzed. A protein-protein interaction (PPI) network was constructed. Hub genes were identified, and their expression was verified using transcriptome data of mice fed 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and Mdr2-/- mice (GSE179993, GSE80776), as well as by immunohistochemistry staining on clinical samples. The correlations between the key gene and other factors were evaluated by Pearson's correlation coefficient. Immune cell infiltration into human liver (GSE159676) was analyzed by xCell and verified by immunofluorescence staining on PSC liver samples. RESULTS: Of the 185 DEGs identified, 113 were upregulated and 72 were downregulated genes in PSC. Genes associated with immune cell infiltration and fibrosis were significantly enriched in PSC. PPI network showed close interactions among DEGs. A module strongly associated with immune infiltration was identified, with annexin A1 (ANXA1) being the core gene. High expression of ANXA1 in PSC was confirmed in two public datasets and by immunohistochemistry staining on clinical samples. High ANXA1 expression was strongly associated with high-risk score for PSC. Also, ANXA1 expression was positively associated with chemokines and chemokine receptors and with the infiltration of immune cells, especially T cells, into liver with PSC. Immune infiltration, fibrosis, and cancer-related processes were markedly enriched in PSC with high expression of ANXA1. CONCLUSION: ANXA1 is a key gene associated with high risk and infiltration of immune cells, especially T cells, in PSC. These findings provide new insight into the key biomarker of PSC and suggest that targeting ANXA1 may be a valuable strategy for the treatment of PSC.

Association between autoimmune liver diseases and chronic hepatitis B: A multivariable Mendelian randomization study in European population.

BACKGROUND: Observational studies have indicated a link between autoimmune liver diseases (AILD) and chronic hepatitis B (CHB) through observational studies. The association between AILD and CHB remains indeterminate. METHODS: A two-sample Mendelian randomization (MR) analysis was conducted to scrutinize the causal nexus between AILD and CHB utilizing summary statistics derived from extensive genome-wide association studies (GWASs) in European populations. The primary statistical methodology employed was the inverse variance-weighted (IVW) method to deduce the causal connection of AILD on CHB. This study incorporated primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), and autoimmune hepatitis (AIH) as subtypes of AILD. Additionally, we conducted a multivariable MR (MVMR) analysis to account for the potential confounding effects of smoking, alcohol consumption, body mass index (BMI), and some autoimmune diseases. RESULTS: Our MR investigation encompassed a cohort of 725,816 individuals. The MR analysis revealed that genetically predicted PSC significantly correlated with a reduced risk of CHB (IVW OR = 0.857; 95%CI: 0.770-0.953, P = 0.005). Conversely, the reverse MR analysis suggested that genetic susceptibility to PSC might not modify the risk of CHB (IVW OR = 1.004; 95% CI: 0.958-1.053, P = 0.866). Genetically proxied PBC and AIH exhibited no discernible causal association with CHB in the MR analysis using the IVW method (P = 0.583; P = 0.425). The MVMR analysis still indicated a decreased risk of CHB associated with PSC (OR = 0.853, P = 0.003). CONCLUSION: Our study elucidates a causal relationship between PSC and a diminished risk of CHB.

Investigating the shared genetic architecture between primary sclerosing cholangitis and inflammatory bowel diseases: a Mendelian randomization study.

BACKGROUND: Several studies have found that primary sclerosing cholangitis (PSC) and inflammatory bowel disease (IBD) are closely associated. However, the direction and causality of their interactions remain unclear. Thus, this study employs Mendelian Randomization to explore whether there are causal associations of genetically predicted PSC with IBD. METHODS: Genetic variants associated with the genome-wide association study (GWAS) of PSC were used as instrumental variables. The statistics for IBD, including ulcerative colitis (UC), and Crohn's disease (CD) were derived from GWAS. Then, five methods were used to estimate the effects of genetically predicted PSC on IBD, including MR Egger, Weighted median (WM), Inverse variance weighted (IVW), Simple mode, and Weighted mode. Last, we also evaluated the pleiotropic effects, heterogeneity, and a leave-one-out sensitivity analysis that drives causal associations to confirm the validity of the analysis. RESULTS: Genetically predicted PSC was significantly associated with an increased risk of UC, according to the study (odds ratio [OR] IVW= 1.0014, P<0.05). However, none of the MR methods found significant causal evidence of genetically predicted PSC in CD (All P>0.05). The sensitivity analysis results showed that the causal effect estimations of genetically predicted PSC on IBD were robust, and there was no horizontal pleiotropy or statistical heterogeneity. CONCLUSIONS: Our study corroborated a causal association between genetically predicted PSC and UC but did not between genetically predicted PSC and CD. Then, we identification of shared SNPs for PSC and UC, including rs3184504, rs9858213, rs725613, rs10909839, and rs4147359. More animal experiments and clinical observational studies are required to further clarify the underlying mechanisms of PSC and IBD.

Genetically predicted gut microbiota mediate the association between plasma lipidomics and primary sclerosing cholangitis.

BACKGROUND: Primary sclerosing cholangitis (PSC) is a complex disease with pathogenic mechanisms that remain to be elucidated. Previous observational studies with small sample sizes have reported associations between PSC, dyslipidemia, and gut microbiota dysbiosis. However, the causality of these associations is uncertain, and there has been no systematic analysis to date. METHODS: The datasets comprise data on PSC, 179 lipid species, and 412 gut microbiota species. PSC data (n = 14,890) were sourced from the International PSC Study Group, while the dataset pertaining to plasma lipidomics originated from a study involving 7174 Finnish individuals. Data on gut microbiota species were derived from the Dutch Microbiome Project study, which conducted a genome-wide association study involving 7738 participants. Furthermore, we employed a two-step Mendelian randomization (MR) analysis to quantify the proportion of the effect of gut microbiota-mediated lipidomics on PSC. RESULTS: Following a rigorous screening process, our MR analysis revealed a causal relationship between higher levels of gene-predicted Phosphatidylcholine (O-16:1_18:1) (PC O-16:1_18:1) and an increased risk of developing PSC (inverse variance-weighted method, odds ratio (OR) 1.30, 95% confidence interval (CI) 1.03-1.63). There is insufficient evidence to suggest that gene-predicted PSC impacts the levels of PC O-16:1_18:1 (OR 1.01, 95% CI 0.98-1.05). When incorporating gut microbiota data into the analysis, we found that Eubacterium rectale-mediated genetic prediction explains 17.59% of the variance in PC O-16:1_18:1 levels. CONCLUSION: Our study revealed a causal association between PC O-16:1_18:1 levels and PSC, with a minor portion of the effect mediated by Eubacterium rectale. This study aims to further explore the pathogenesis of PSC and identify promising therapeutic targets. For patients with PSC who lack effective treatment options, the results are encouraging.

Primary sclerosing cholangitis causally affects kidney function decline: A Mendelian randomization study.

BACKGROUND AND AIM: The causal linkage between primary sclerosing cholangitis (PSC) and kidney function is unexplored despite their potential for long-term detrimental effects on kidney function. METHODS: Two-sample summary-level Mendelian randomization (MR) study was conducted to identify the association between PSC and kidney function. The genetic variants were extracted from the PSC-specific multi-trait analyzed genome-wide association study (GWAS) of European ancestry. Summary-level data for kidney function traits, including estimated glomerular filtration rate (eGFR), annual eGFR decline, and chronic kidney disease (CKD), were obtained from the CKDGen consortium. Multiplicative random-effects inverse-variance weighted (MR-IVW), and a series of pleiotropy-robust analyses were performed to investigate the causal effects and ascertain their robustness. RESULTS: Significant causal associations between genetically predicted PSC and kidney function traits were identified. Genetically predicted PSC was associated with decreased log-transformed eGFR (MR-IVW; beta = -0.41%; standard error [SE] = 0.02%; P < 0.001), increased rate of annual eGFR decline (MR-IVW; beta = 2.43%; SE = 0.18%; P < 0.001), and higher risk of CKD (MR-IVW; odds ratio = 1.07; 95% confidence interval = 1.06-1.08; P < 0.001). The main findings were supported by pleiotropy-robust analysis, including MR-Egger with bootstrapped error and weighted median. CONCLUSIONS: Our study demonstrates that genetically predicted PSC is causally associated with kidney function impairment. Further studies are warranted to identify the underlying mechanisms.

Loss of apical sodium bile acid transporter alters bile acid circulation and reduces biliary damage in cholangitis.

Primary sclerosing cholangitis (PSC) is characterized by increased ductular reaction (DR), liver fibrosis, hepatic total bile acid (TBA) levels, and mast cell (MC) infiltration. Apical sodium BA transporter (ASBT) expression increases in cholestasis, and ileal inhibition reduces PSC phenotypes. FVB/NJ and multidrug-resistant 2 knockout (Mdr2-/-) mice were treated with control or ASBT Vivo-Morpholino (VM). We measured 1) ASBT expression and MC presence in liver/ileum; 2) liver damage/DR; 3) hepatic fibrosis/inflammation; 4) biliary inflammation/histamine serum content; and 5) gut barrier integrity/hepatic bacterial translocation. TBA/BA composition was measured in cholangiocyte/hepatocyte supernatants, intestine, liver, serum, and feces. Shotgun analysis was performed to ascertain microbiome changes. In vitro, cholangiocytes were treated with BAs ± ASBT VM, and histamine content and farnesoid X receptor (FXR) signaling were determined. Treated cholangiocytes were cocultured with MCs, and FXR signaling, inflammation, and MC activation were measured. Human patients were evaluated for ASBT/MC expression and histamine/TBA content in bile. Control patient- and PSC patient-derived three-dimensional (3-D) organoids were generated; ASBT, chymase, histamine, and fibroblast growth factor-19 (FGF19) were evaluated. ASBT VM in Mdr2-/- mice decreased 1) biliary ASBT expression, 2) PSC phenotypes, 3) hepatic TBA, and 4) gut barrier integrity compared with control. We found alterations between wild-type (WT) and Mdr2-/- mouse microbiome, and ASBT/MC and bile histamine content increased in cholestatic patients. BA-stimulated cholangiocytes increased MC activation/FXR signaling via ASBT, and human PSC-derived 3-D organoids secrete histamine/FGF19. Inhibition of hepatic ASBT ameliorates cholestatic phenotypes by reducing cholehepatic BA signaling, biliary inflammation, and histamine levels. ASBT regulation of hepatic BA signaling offers a therapeutic avenue for PSC.NEW & NOTEWORTHY We evaluated knockdown of the apical sodium bile acid transporter (ASBT) using Vivo-Morpholino in Mdr2KO mice. ASBT inhibition decreases primary sclerosing cholangitis (PSC) pathogenesis by reducing hepatic mast cell infiltration, altering bile acid species/cholehepatic shunt, and regulating gut inflammation/dysbiosis. Since a large cohort of PSC patients present with IBD, this study is clinically important. We validated findings in human PSC and PSC-IBD along with studies in novel human 3-D organoids formed from human PSC livers.

Early and accurate detection of cholangiocarcinoma in patients with primary sclerosing cholangitis by methylation markers in bile.

BACKGROUND AND AIMS: Primary sclerosing cholangitis (PSC) is associated with increased risk of cholangiocarcinoma (CCA). Early and accurate CCA detection represents an unmet clinical need as the majority of patients with PSC are diagnosed at an advanced stage of malignancy. In the present study, we aimed at establishing robust DNA methylation biomarkers in bile for early and accurate diagnosis of CCA in PSC. APPROACH AND RESULTS: Droplet digital PCR (ddPCR) was used to analyze 344 bile samples from 273 patients with sporadic and PSC-associated CCA, PSC, and other nonmalignant liver diseases for promoter methylation of cysteine dioxygenase type 1, cannabinoid receptor interacting protein 1, septin 9, and vimentin. Receiver operating characteristic (ROC) curve analyses revealed high AUCs for all four markers (0.77-0.87) for CCA detection among patients with PSC. Including only samples from patients with PSC diagnosed with CCA ≤ 12 months following bile collection increased the accuracy for cancer detection, with a combined sensitivity of 100% (28/28) and a specificity of 90% (20/203). The specificity increased to 93% when only including patients with PSC with longtime follow-up (> 36 months) as controls, and remained high (83%) when only including patients with PSC and dysplasia as controls (n = 23). Importantly, the bile samples from the CCA-PSC ≤ 12 patients, all positive for the biomarkers, included both early-stage and late-stage CCA, different tumor growth patterns, anatomical locations, and carbohydrate antigen 19-9 levels. CONCLUSIONS: Using highly sensitive ddPCR to analyze robust epigenetic biomarkers, CCA in PSC was accurately detected in bile, irrespective of clinical and molecular features, up to 12 months before CCA diagnosis. The findings suggest a potential for these biomarkers to complement current detection and screening methods for CCA in patients with PSC.

Analysis of various ATP-binding cassette transporters revealed quantification of ABCB4 as a potential diagnostic tool in primary sclerosing cholangitis (PSC).

AIMS: ATP-binding cassette transporters are important proteins in regulating bile constituent transport between hepatocytes and the bile canalicular system. Dysfunctional transporters lead to accumulation of toxic bile components within hepatocytes or the biliary system, known as cholestasis, resulting in liver damage. It has been previously reported that two particular ATP-binding cassette transporters, ABCB4 and ABCB11, have altered expression in patients with primary sclerosing cholangitis (PSC). Interested in further analysis of expression patterns of ATP-binding cassette transporters in PSC patients, we investigated liver samples from 201 patients, including 43 patients with PSC and 51 patients with primary biliary cholangitis patients (PBC). In addition to ABCB4 and ABCB11, we also included other ATP-binding cassette transporters, to determine if upregulation of ABCB4 and ABCB11 is specifically found in the liver of patients with PSC. METHODS AND RESULTS: Retrospectively, formalin-fixed and paraffin-embedded liver biopsies, resections, and explants were selected to investigate the expression of ABCB1, ABCB4, ABCB11, ABCG5/8, and FXR1 using nanoString nCounter and immunohistochemistry for validation of differently expressed transporters seen in PSC liver samples in comparison to non-PSC liver specimens. Strikingly, ABCB4 was the only ATP-binding cassette transporter showing increased gene and protein expression in hepatocytes of PSC livers when compared to non-PSC liver specimens. Furthermore, ABCB4 protein expression also correlated with disease stage in PSC. CONCLUSION: Our study concluded that altered ABCB4 expression is specifically seen in liver specimens of PSC patients. Therefore, quantitative ABCB4 analysis may be an additional useful tool for the histopathological diagnosis of PSC to distinguish this entity from other cholangiopathies.

Next-generation sequencing mutation analysis on biliary brush cytology for differentiation of benign and malignant strictures in primary sclerosing cholangitis.

BACKGROUND AND AIMS: Differentiation of benign and malignant biliary tract strictures on brush material remains highly challenging but is essential for adequate clinical management of patients with primary sclerosing cholangitis (PSC). In this case-control study, biliary brush cytology samples from PSC patients with cholangiocarcinoma (PSC-CCA) were compared with samples from PSC patients without CCA (PSC-control subjects) using next-generation sequencing (NGS). METHODS: Cells on archived slides were dissected for DNA extraction. NGS was performed using a gene panel containing 242 hotspots in 14 genes. Repeated brush samples from the same patient were analyzed to study the consistency of NGS results. In PSC-CCA cases that underwent surgical resection, molecular aberrations in brush samples were compared with NGS data from subsequent resection specimens. RESULTS: Forty patients (20 PSC-CCA and 20 PSC-control subjects) were included. The gene panel detected 22 mutations in 15 of 20 PSC-CCA brush samples, including mutations in TP53 (8 brush samples), K-ras (5), G-nas (3), ERBB2 (1), APC (1), PIK3CA (1), and SMAD4 (1). One G-nas and 3 K-ras mutations were found in 3 of 20 PSC-control brush samples. The sensitivity of the NGS panel was 75% (95% confidence interval, 62%-80%) and specificity 85% (95% confidence interval, 64%-95%). Repeated brush samples showed identical mutations in 6 of 9 cases. Three repeated brush samples demonstrated additional mutations as compared with the first brush sample. In 6 of 7 patients, mutations in brush samples were identical to mutations in subsequent resection specimens. CONCLUSIONS: NGS mutation analysis of PSC brush cytology detects oncogenic mutations with high sensitivity and specificity and seems to constitute a valuable adjunct to cytologic assessment of brush samples.

Biallelic known and novel DCDC2 variants in cholestatic liver disease: Phenotype-genotype observations in four children.

Neonatal sclerosing cholangitis (NSC) is associated with progressing biliary fibrosis that often requires liver transplantation in childhood. Several recent studies have identified variants in DCDC2, encoding doublecortin domain-containing protein 2 (DCDC2), expressed in primary cilia, that accompany syndromic disease and NSC. We report four patients with hepatobiliary disease associated with two novel homozygous or compound heterozygous variants in DCDC2. Three patients with protein-truncating variants in DCDC2, expressing no DCDC2, presented with the originally described severe hepatic phenotype in infancy. One patient with a novel homozygous DCDC2 missense variant shows a markedly milder phenotype only manifest in childhood and with retained DCDC2 expression. Concomitant nephronophthisis is present in three patients and learning disability in two. This report widens the phenotypic spectrum of DCDC2-associated hepatobiliary disease. Testing for DCDC2 expression and DCDC2 variants should be included in the evaluation of cholangiopathy of unknown aetiology in childhood as well as in infancy.

Genetic alterations during the neoplastic cascade towards cholangiocarcinoma in primary sclerosing cholangitis.

Carcinogenesis of primary sclerosing cholangitis (PSC)-associated cholangiocarcinoma (CCA) is largely unexplored. Improved understanding of the molecular events involved may guide development of novel avenues for rational clinical management. We aimed to assess the genetic alterations during progression of the neoplastic cascade from biliary dysplasia towards CCA in PSC. Forty-four resection specimens or biopsies of PSC patients with biliary dysplasia (n = 2) and/or CCA (n = 42) were included. DNA was extracted from sections of formalin-fixed paraffin-embedded tissue blocks with dysplasia (n = 23), CCA (n = 69), and nonneoplastic tissue (n = 28). A custom-made next-generation sequencing (NGS) panel of 28 genes was used for mutation and copy number variation (CNV) detection. In addition, CNVs of CDKN2A, EGFR, MCL1, and MYC were examined by fluorescence in situ hybridization. Alterations in 16 low-grade dysplasia samples included loss of FGFR1 (19%), CDKN2A (13%), and SMAD4 (6%), amplification of FGFR3 (6%), EGFR (6%), and ERBB2 (6%), and mutations in SMAD4 (13%). High-grade dysplasia (n = 7) is characterized by MYC amplification (43%), and mutations in ERBB2 (71%) and TP53 (86%). TP53 mutations are the most common aberrations in PSC-CCA (30%), whereas mutations in KRAS (16%), GNAS (14%), and PIK3CA (9%) are also common. In conclusion, PSC-CCA exhibits a variety of genetic alterations during progression of the neoplastic cascade, with mainly CNVs being present early, whereas mutations in ERBB2, TP53, and KRAS appear later in the development of CCA. These findings are promising for the development of NGS-guided diagnostic strategies in PSC-CCA. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The Role of hsa-miR-125b-5p Interaction with S1P/Ceramide Axis in the Potential Development of Inflammation-Associated Colon Cancer in Primary Sclerosing Cholangitis.

Primary sclerosing cholangitis (PSC) is characterised by the co-occurrence of inflammatory bowel diseases, particularly ulcerative colitis (UC). We investigated how the interaction of miR-125b with the sphingosine-1-phosphate (S1P)/ceramide axis may predispose patients with PSC, PSC/UC, and UC to carcinogenesis in the ascending and sigmoid colons. The overexpression of miR-125b was accompanied by the upregulation of S1P, ceramide synthases, ceramide kinases, and the downregulation of AT-rich interaction domain 2 in the ascending colon of PSC/UC, which contributed to the progression of high microsatellite instability (MSI-H) colorectal carcinoma. We also showed that the overexpression of sphingosine kinase 2 (SPHK2) and the genes involved in the glycolytic pathway in the sigmoid colon of UC led to the upregulation of Interleukin 17 (IL-17). In vitro stimulation of human intestinal epithelial cells (Caco-2, HT-29, and NCM460D) with lipopolysaccharide suppressed miR-125b and increased proinflammatory cytokines, whereas the induction of miR-125b activity by either a miR-125b mimetic or lithocholic acid resulted in the inhibition of miR-125b targets. In summary, miR-125b overexpression was associated with an imbalance in the S1P/ceramide axis that can lead to MSI-H cancer progression in PSC/UC. Furthermore, SPHK2 overexpression and a change in the cellular metabolic flux are important players in inflammation-associated colon cancer in UC.

Activation of the GPR35 pathway drives angiogenesis in the tumour microenvironment.

OBJECTIVE: Primary sclerosing cholangitis (PSC) is in 70% of cases associated with inflammatory bowel disease. The hypermorphic T108M variant of the orphan G protein-coupled receptor GPR35 increases risk for PSC and ulcerative colitis (UC), conditions strongly predisposing for inflammation-associated liver and colon cancer. Lack of GPR35 reduces tumour numbers in mouse models of spontaneous and colitis associated cancer. The tumour microenvironment substantially determines tumour growth, and tumour-associated macrophages are crucial for neovascularisation. We aim to understand the role of the GPR35 pathway in the tumour microenvironment of spontaneous and colitis-associated colon cancers. DESIGN: Mice lacking GPR35 on their macrophages underwent models of spontaneous colon cancer or colitis-associated cancer. The role of tumour-associated macrophages was then assessed in biochemical and functional assays. RESULTS: Here, we show that GPR35 on macrophages is a potent amplifier of tumour growth by stimulating neoangiogenesis and tumour tissue remodelling. Deletion of Gpr35 in macrophages profoundly reduces tumour growth in inflammation-associated and spontaneous tumour models caused by mutant tumour suppressor adenomatous polyposis coli. Neoangiogenesis and matrix metalloproteinase activity is promoted by GPR35 via Na/K-ATPase-dependent ion pumping and Src activation, and is selectively inhibited by a GPR35-specific pepducin. Supernatants from human inducible-pluripotent-stem-cell derived macrophages carrying the UC and PSC risk variant stimulate tube formation by enhancing the release of angiogenic factors. CONCLUSIONS: Activation of the GPR35 pathway promotes tumour growth via two separate routes, by directly augmenting proliferation in epithelial cells that express the receptor, and by coordinating macrophages' ability to create a tumour-permissive environment.

Human Leukocyte Antigen Profile Predicts Severity of Autoimmune Liver Disease in Children of European Ancestry.

BACKGROUND AND AIMS: Genetic predisposition to autoimmune hepatitis (AIH) in adults is associated with possession of human leukocyte antigen (HLA) class I (A*01, B*08) and class II (DRB1*03, -04, -07, or -13) alleles, depending on geographic region. Juvenile autoimmune liver disease (AILD) comprises AIH-1, AIH-2, and autoimmune sclerosing cholangitis (ASC), which are phenotypically different from their adult counterparts. We aimed to define the relationship between HLA profile and disease course, severity, and outcome in juvenile AILD. APPROACH AND RESULTS: We studied 236 children of European ancestry (152 female [64%], median age 11.15 years, range 0.8-17), including 100 with AIH-1, 59 with AIH-2, and 77 with ASC. The follow-up period was from 1977 to June 2019 (median 14.5 years). Class I and II HLA genotyping was performed using PCR/sequence-specific primers. HLA B*08, -DRB1*03, and the A1-B8-DR3 haplotype impart predisposition to all three forms of AILD. Homozygosity for DRB1*03 represented the strongest risk factor (8.8). HLA DRB1*04, which independently confers susceptibility to AIH in adults, was infrequent in AIH-1 and ASC, suggesting protection; and DRB1*15 (DR15) was protective against all forms of AILD. Distinct HLA class II alleles predispose to the different subgroups of juvenile AILD: DRB1*03 to AIH-1, DRB1*13 to ASC, and DRB1*07 to AIH-2. Possession of homozygous DRB1*03 or of DRB1*13 is associated with fibrosis at disease onset, and possession of these two genes in addition to DRB1*07 is associated with a more severe disease in all three subgroups. CONCLUSIONS: Unique HLA profiles are seen in each subgroup of juvenile AILD. HLA genotype might be useful in predicting responsiveness to immunosuppressive treatment and course.