Cell-substrate distance fluctuations of confluent cells enable fast and coherent collective migration.

Collective cell migration is an emergent phenomenon, with long-range cell-cell communication influenced by various factors, including transmission of forces, viscoelasticity of individual cells, substrate interactions, and mechanotransduction. We investigate how alterations in cell-substrate distance fluctuations, cell-substrate adhesion, and traction forces impact the average velocity and temporal-spatial correlation of confluent monolayers formed by either wild-type (WT) MDCKII cells or zonula occludens (ZO)-1/2-depleted MDCKII cells (double knockdown [dKD]) representing highly contractile cells. The data indicate that confluent dKD monolayers exhibit decreased average velocity compared to less contractile WT cells concomitant with increased substrate adhesion, reduced traction forces, a more compact shape, diminished cell-cell interactions, and reduced cell-substrate distance fluctuations. Depletion of basal actin and myosin further supports the notion that short-range cell-substrate interactions, particularly fluctuations driven by basal actomyosin, significantly influence the migration speed of the monolayer on a larger length scale.

Genotype correlates with clinical course and outcome of children with tight junction protein 2 (TJP2) deficiency-related cholestasis.

BACKGROUND AND AIMS: The study aimed to describe the clinical course and outcomes, and analyze the genotype-phenotype correlation in patients with tight junction protein 2 (TJP2) deficiency. APPROACH AND RESULTS: Data from all children with chronic cholestasis and either homozygous or compound heterozygous mutations in TJP2 were extracted and analyzed. The patients were categorized into 3 genotypes: TJP2-A (missense mutations on both alleles), TJP2-B (missense mutation on one allele and a predicted protein-truncating mutation [PPTM] on the other), and TJP2-C (PPTMs on both alleles). A total of 278 cases of genetic intrahepatic cholestasis were studied, with TJP2 deficiency accounting for 44 cases (15.8%). Of these, 29 were homozygous and 15 were compound heterozygous variants of TJP2 . TJP2-A genotype was identified in 21 (47.7%), TJP2-B in 7 cases (15.9%), and TJP2-C in 16 cases (36.4%), respectively. Patients with the TJP2-C genotype were more likely to experience early infantile cholestasis (87.5% vs. 53.5%, p =0.033), less likely to clear jaundice (12.5% vs. 52.2%, p =0.037), more likely to develop ascites, and had higher serum bile acids. Patients with the TJP2-C genotype were more likely to die or require liver transplantation (native liver survival: 12.5% vs. 78.6%, p <0.001), with a median age at death/liver transplantation of 2.5 years. Cox regression analysis revealed that TJP2-C mutations ( p =0.003) and failure to resolve jaundice ( p =0.049) were independent predictors of poor outcomes. CONCLUSIONS: Patients with the TJP2-C genotype carrying PPTMs in both alleles had a rapidly progressive course, leading to early decompensation and death if they did not receive timely liver transplantation.

ZNF582 overexpression restrains the progression of clear cell renal cell carcinoma by enhancing the binding of TJP2 and ERK2 and inhibiting ERK2 phosphorylation.

Recent evidences have suggested that Zinc finger protein 582 (ZNF582) plays different important roles in various tumors, but its clinical role, biological function and regulatory mechanism in clear cell renal cell carcinoma (ccRCC) are still vague. Through analyzing GEO and TCGA-KIRC data and validation with local samples, we identified the low expression pattern of ZNF582 in ccRCC. Decreased ZNF582 expression is correlated with higher tumor stage and grade, distant metastasis and poor prognosis. By analyzing the DNA methylation data of ccRCC in TCGA-KIRC and using Massarray DNA methylation and demethylation analysis, we confirmed the hypermethylation status of ZNF582 in ccRCC and its negative regulation on ZNF582 expression. Using cell phenotype experiments and orthotopic kidney tumor growth models, we determined the inhibitory effect of ZNF582 overexpression on ccRCC growth and metastasis in vivo and in vitro. Mechanistically, using TMT (Tandem mass tags) quantitative proteomics test, Co-IP (Co-immunoprecipitation) and Western Blot experiments, we clarified that ZNF582 binds to TJP2 and up-regulates TJP2 protein expression. Increased TJP2 protein combines with ERK2 to promote ERK2 protein expression and suppresses the phosphorylation of ERK2, thereby inhibiting the growth and metastasis of ccRCC. In general, our findings provide the first solid theoretical rationale for targeting ZNF582/TJP2/ERK2 axis to improve ccRCC treatment.

MAGIs regulate aPKC to enable balanced distribution of intercellular tension for epithelial sheet homeostasis.

Constriction of the apical plasma membrane is a hallmark of epithelial cells that underlies cell shape changes in tissue morphogenesis and maintenance of tissue integrity in homeostasis. Contractile force is exerted by a cortical actomyosin network that is anchored to the plasma membrane by the apical junctional complexes (AJC). In this study, we present evidence that MAGI proteins, structural components of AJC whose function remained unclear, regulate apical constriction of epithelial cells through the Par polarity proteins. We reveal that MAGIs are required to uniformly distribute Partitioning defective-3 (Par-3) at AJC of cells throughout the epithelial monolayer. MAGIs recruit ankyrin-repeat-, SH3-domain- and proline-rich-region-containing protein 2 (ASPP2) to AJC, which modulates Par-3-aPKC to antagonize ROCK-driven contractility. By coupling the adhesion machinery to the polarity proteins to regulate cellular contractility, we propose that MAGIs play essential and central roles in maintaining steady state intercellular tension throughout the epithelial cell sheet.

Protective Functions of ZO-2/Tjp2 Expressed in Hepatocytes and Cholangiocytes Against Liver Injury and Cholestasis.

BACKGROUND & AIMS: Liver tight junctions (TJs) establish tissue barriers that isolate bile from the blood circulation. TJP2/ZO-2-inactivating mutations cause progressive cholestatic liver disease in humans. Because the underlying mechanisms remain elusive, we characterized mice with liver-specific inactivation of Tjp2. METHODS: Tjp2 was deleted in hepatocytes, cholangiocytes, or both. Effects on the liver were assessed by biochemical analyses of plasma, liver, and bile and by electron microscopy, histology, and immunostaining. TJ barrier permeability was evaluated using fluorescein isothiocyanate-dextran (4 kDa). Cholic acid (CA) diet was used to assess susceptibility to liver injury. RESULTS: Liver-specific deletion of Tjp2 resulted in lower Cldn1 protein levels, minor changes to the TJ, dilated canaliculi, lower microvilli density, and aberrant radixin and bile salt export pump (BSEP) distribution, without an overt increase in TJ permeability. Hepatic Tjp2-defcient mice presented with mild progressive cholestasis with lower expression levels of bile acid transporter Abcb11/Bsep and detoxification enzyme Cyp2b10. A CA diet tolerated by control mice caused severe cholestasis and liver necrosis in Tjp2-deficient animals. 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene ameliorated CA-induced injury by enhancing Cyp2b10 expression, and ursodeoxycholic acid provided partial improvement. Inactivating Tjp2 separately in hepatocytes or cholangiocytes showed only mild CA-induced liver injury. CONCLUSION: Tjp2 is required for normal cortical distribution of radixin, canalicular volume regulation, and microvilli density. Its inactivation deregulated expression of Cldn1 and key bile acid transporters and detoxification enzymes. The mice provide a novel animal model for cholestatic liver disease caused by TJP2-inactivating mutations in humans.

The zonula occludens protein family regulates the hepatic barrier system in the murine liver.

The hepatic barrier is indispensable for the physiological functions of the liver and is impaired under various pathological conditions. Tight junctions reportedly play a central role in hepatic barrier regulation; however, there is limited direct evidence supporting this observation, with few in vivo models or confirmations of the implicated molecular mechanisms presented to date. We inactivated the tight junction component gene, Tjp2/ZO-2, and the related molecule, Tjp1/ZO-1, in mouse livers. In humans, TJP2/ZO-2 mutations have been implicated in the development of human progressive familial intrahepatic cholestasis 4 (PFIC4). The mice deficient in either ZO-1 or ZO-2 in the liver did not exhibit major abnormalities. However, the ablation of both molecules impaired the molecular architecture as well as the structure and function of hepatocyte tight junctions, which disrupted the hepatic barrier and was lethal to the mice by 6 weeks of age. In mutant mice, bile canaliculus formation and cellular polarity were compromised; also, transporter expression and localization were deregulated. Moreover, typical hepatic zonation and bile duct formation were inhibited, and sinusoidal vessels were disorganized. These findings clarify the role of tight junctions and polarity in the hepatic barrier as well as the effect that their disruption has on liver tissue. The observations also suggest that liver-specific ZO-1-/- and ZO-2-/- mice could be used as models for PFIC4, and this will provide new insights into liver pathophysiology and clinical applications.

Progressive familial intrahepatic cholestasis type 4 in an Indian child: presentation, initial course and novel compound heterozygous mutation.

A 15-year-old boy who had a history of on and off pruritus and jaundice since many years found to have a novel mutation in TJP2 gene. On examination, he had clubbing, splenomegaly, grade 3 oesophageal varices and short stature. Investigation revealed direct hyperbirubinemia with elevated liver enzymes with normal gamma-glutamyl transferase (GGT). Antinuclear antibody (ANA), smooth muscle antibody (SMA) anti-liver kidney microsomal (anti-LKM) and viral markers for hepatitis were negative. However, IgG was elevated and anti-smooth muscle antibody (ASMA) was weekly positive (1:20). He was also given a trial of steroid and azathioprine for 1 year on the basis of liver biopsy findings, raised IgG and positive ASMA but finding no improvement stopped. Genetic testing by next-generation sequencing found a novel compound heterozygous missense variation in exon 17 of the TJP2 gene confirming progressive familial intrahepatic cholestasis type 4 as the aetiology of cholestatic liver disease.

New tight junction protein 2 variant causing progressive familial intrahepatic cholestasis type 4 in adults: A case report.

BACKGROUND: Progressive familial intrahepatic cholestasis (PFIC) encompasses a group of autosomal recessive disorders with high morbidity and mortality. Variants in the gene encoding tight junction protein-2 (TJP2) have been linked to PFIC type 4 (PFIC4), which predominantly presents in childhood. However, there are only limited data from adults with TJP2-related PFIC4. We report a family with an autosomal recessive disorder with a novel variant in the TJP2 gene in adults with very variable expression of PFIC4. CASE SUMMARY: The index patient presented at 19 years old with liver cirrhosis and variceal bleeding and was treated with endoscopic banding and beta-blockers. In 2018, he developed primary liver cancer that was treated with radiofrequency ablation followed by liver transplantation in 2019. Genetic testing revealed a novel homozygous TJP2 variant causing PFIC4 (TJP2([NM_004817.3]:c.[3334C>T]; [3334C>T])). The consanguineous family consists of the father and mother (both heterozygous) and their 12 children, of which five carry the variant in a homozygous state; however, these five siblings have highly variable expression of PFIC4. Two homozygous brothers had cirrhosis and portal hypertension at diagnosis at the ages of 19 and 36. Two other homozygous brothers, age 23 and 19, and the homozygous sister, age 21, have elevated liver enzymes but presently no cirrhosis, which may suggest an age-dependent penetrance. In addition, five sisters had severe and mild intrahepatic cholestasis of pregnancy and carry the TJP2 variant in a homozygous and heterozygous state, respectively. CONCLUSION: This novel TJP2 variant is associated with PFIC4 causing severe liver disease with cirrhosis and primary liver cancer in adolescents/adults.

Podosome formation impairs endothelial barrier function by sequestering zonula occludens proteins.

Podosomes and tight junctions (TJs) are subcellular compartments that both exist in endothelial cells and localize at cell surfaces. In contrast to the well-characterized role of TJs in maintaining cerebrovascular integrity, the specific function of endothelial podosomes remains unknown. Intriguingly, we discovered cross-talk between podosomes and TJs in human brain endothelial cells. Tight junction scaffold proteins ZO-1 and ZO-2 localize at podosomes in response to phorbol-12-myristate-13-acetate treatment. We found that both ZO proteins are essential for podosome formation and function. Rather than being derived from new protein synthesis, podosomal ZO-1 and ZO-2 are relocated from a pre-existing pool found at the peripheral plasma membrane with enhanced physical interaction with cortactin, a known protein marker for podosomes. Sequestration of ZO proteins in podosomes weakens tight junction complex formation, leading to increased endothelial cell permeability. This effect can be further attenuated by podosome inhibitor PP2. Altogether, our data revealed a novel cellular function of podosomes, specifically, their ability to negatively regulate tight junction and endothelial barrier integrity, which have been linked to a variety of cerebrovascular diseases.

TJP2 hepatobiliary disorders: Novel variants and clinical diversity.

To assess the spectrum of pediatric clinical phenotypes in TJP2 disease, we reviewed records of our seven patients in whom intrahepatic cholestasis was associated with biallelic TJP2 variants (13; 12 novel) and correlated clinical manifestations with mutation type. The effect of a splicing variant was analyzed with a minigene assay. The effects of three missense variants were analyzed with protein expression in vitro. Our patients had both remitting and persistent cholestasis. Three exhibited growth retardation. Six responded to treatment with cholestyramine, ursodeoxycholic acid, or both. Two had cholecystolithiasis. None required liver transplantation or developed hepatocellular or cholangiocellular malignancy. None manifested extrahepatic disease not attributable to effects of cholestasis. The variant c.2180-5T>G resulted in exon 15 skipping with in-frame deletion of 32 amino acid residues in TJP2. The three missense variants decreased but did not abolish TJP2 expression. Patients with truncating or canonical splice-site variants had clinically more severe disease. TJP2 disease in children includes a full clinical spectrum of severity, with mild or intermittent forms as well as the severe and minimal forms hitherto described. Biallelic TJP2 variants must be considered in children with clinically intermittent or resolved intrahepatic cholestasis.

Phase Separation of Zonula Occludens Proteins Drives Formation of Tight Junctions.

Tight junctions are cell-adhesion complexes that seal tissues and are involved in cell polarity and signaling. Supra-molecular assembly and positioning of tight junctions as continuous networks of adhesion strands are dependent on the membrane-associated scaffolding proteins ZO1 and ZO2. To understand how zona occludens (ZO) proteins organize junction assembly, we performed quantitative cell biology and in vitro reconstitution experiments. We discovered that ZO proteins self-organize membrane-attached compartments via phase separation. We identified the multivalent interactions of the conserved PDZ-SH3-GuK supra-domain as the driver of phase separation. These interactions are regulated by phosphorylation and intra-molecular binding. Formation of condensed ZO protein compartments is sufficient to specifically enrich and localize tight-junction proteins, including adhesion receptors, cytoskeletal adapters, and transcription factors. Our results suggest that an active-phase transition of ZO proteins into a condensed membrane-bound compartment drives claudin polymerization and coalescence of a continuous tight-junction belt.

ZO-2 Is a Master Regulator of Gene Expression, Cell Proliferation, Cytoarchitecture, and Cell Size.

ZO-2 is a cytoplasmic protein of tight junctions (TJs). Here, we describe ZO-2 involvement in the formation of the apical junctional complex during early development and in TJ biogenesis in epithelial cultured cells. ZO-2 acts as a scaffold for the polymerization of claudins at TJs and plays a unique role in the blood-testis barrier, as well as at TJs of the human liver and the inner ear. ZO-2 movement between the cytoplasm and nucleus is regulated by nuclear localization and exportation signals and post-translation modifications, while ZO-2 arrival at the cell border is triggered by activation of calcium sensing receptors and corresponding downstream signaling. Depending on its location, ZO-2 associates with junctional proteins and the actomyosin cytoskeleton or a variety of nuclear proteins, playing a role as a transcriptional repressor that leads to inhibition of cell proliferation and transformation. ZO-2 regulates cell architecture through modulation of Rho proteins and its absence induces hypertrophy due to inactivation of the Hippo pathway and activation of mTOR and S6K. The interaction of ZO-2 with viral oncoproteins and kinases and its silencing in diverse carcinomas reinforce the view of ZO-2 as a tumor regulator protein.

ZO-2 Suppresses Cell Migration Mediated by a Reduction in Matrix Metalloproteinase 2 in Claudin-18-Expressing Lung Adenocarcinoma A549 Cells.

Abnormal expression of the tight junctional components claudins (CLDNs) is observed in various malignant tissues. We reported recently that CLDN18 expression is down-regulated in human lung adenocarcinoma tissues. In the present study, we investigated the biological functions of CLDN18 using lung adenocarcinoma A549 cells. Microarray analysis showed that CLDN18 increases zonula occludens (ZO)-2 expression in A549 cells. The ectopic expression of CLDN18 increased nuclear ZO-2 levels, which were inhibited by N-[2-[[3-(4-bromophenyl)-2-propen-1-yl]amino]ethyl]5-isoquinolinesulfonamide (H-89), a nonspecific protein kinase A (PKA) inhibitor, but not by a PKA inhibitor 14-22 amide. In addition, dibutyryl cyclic adenosine monophosphate, an analogue of PKA, did not increase ZO-2 levels. These results suggest that H-89 sensitive factors without PKA are involved in the CLDN18-induced elevation of ZO-2. The cell cycle was affected by neither ZO-2 knockdown in CLDN18-expresssing A549 (CLDN18/A549) cells nor ZO-2 overexpression in A549 cells, suggesting that ZO-2 does not play an important role in the regulation of cell proliferation. The introduction of ZO-2 small interfering RNA (siRNA) into CLDN18/A549 cells increased migration, the expression and activity of matrix metalloproteinase 2 (MMP2), and the reporter activity of an MMP2 promoter construct. Furthermore, H-89 enhanced both mRNA levels and reporter activity of MMP2 in CLDN18/A549 cells. These results suggested that a reduction in CLDN18-dependent ZO-2 expression enhances MMP2 expression in lung adenocarcinoma cells, resulting in the promotion of the cell migration. CLDN18 may be a novel marker for metastasis in lung adenocarcinoma.

Novel compound heterozygote mutations of TJP2 in a Chinese child with progressive cholestatic liver disease.

BACKGROUND: Progressive familial intrahepatic cholestasis (PFIC) is a group of genetic autosomal recessive disorders that predominantly affects young children and results in early-onset progressive liver damage. Several types of PFIC were defined based on different genetic aetiologies in last decades. CASE PRESENTATION: Here, we report a Chinese young child diagnosed as PFIC with variants in tight junction protein 2 (TJP2). The patient was affected by a long history of jaundice, pruritus, and failure to thrive. Highly elevated level of serum total bile acid (TBA) and normal levels of gamma-glutamyltransferase (GGT) were observed at hospitalization. The patient's clinical symptoms could be alleviated by administration of ursodeoxycholic acid. Genetic testing by next generation sequencing (NGS) found novel compound heterozygote mutations c.2448 + 1G > C/c.2639delC (p.T880Sfs*12) in TJP2, which were inherited from her mother and father, respectively. Both mutations were predicted to abolish TJP2 protein translation, and neither has previously been identified. CONCLUSION: We report a Chinese female PFIC child with novel compound heterozygous mutations of TJP2. Genetic testing by NGS is valuable in the clinical diagnosis of hereditary liver disease.

The scaffolding protein ZO-1 coordinates actomyosin and epithelial apical specializations in vitro and in vivo.

Polarized epithelia assemble into sheets that compartmentalize organs and generate tissue barriers by integrating apical surfaces into a single, unified structure. This tissue organization is shared across organs, species, and developmental stages. The processes that regulate development and maintenance of apical epithelial surfaces are, however, undefined. Here, using an intestinal epithelial-specific knockout (KO) mouse and cultured epithelial cells, we show that the tight junction scaffolding protein zonula occludens-1 (ZO-1) is essential for development of unified apical surfaces in vivo and in vitro We found that U5 and GuK domains of ZO-1 are necessary for proper apical surface assembly, including organization of microvilli and cortical F-actin; however, direct interactions with F-actin through the ZO-1 actin-binding region (ABR) are not required. ZO-1 lacking the PDZ1 domain, which binds claudins, rescued apical structure in ZO-1-deficient epithelia, but not in cells lacking both ZO-1 and ZO-2, suggesting that heterodimerization with ZO-2 restores PDZ1-dependent ZO-1 interactions that are vital to apical surface organization. Pharmacologic F-actin disruption, myosin II motor inhibition, or dynamin inactivation restored apical epithelial structure in vitro and in vivo, indicating that ZO-1 directs epithelial organization by regulating actomyosin contraction and membrane traffic. We conclude that multiple ZO-1-mediated interactions contribute to coordination of epithelial actomyosin function and genesis of unified apical surfaces.

Progressive Familial Intrahepatic Cholestasis.

Genetic cholestasis has been dissected through genetic investigation. The major PFIC genes are now described. ATP8B1 encodes FIC1, ABCB11 encodes BSEP, ABCB4 encodes MDR3, TJP2 encodes TJP2, NR1H4 encodes FXR, and MYO5B encodes MYO5B. The full spectra of phenotypes associated with mutations in each gene are discussed, along with our understanding of the disease mechanisms. Differences in treatment response and targets for future treatment are emerging.

Effects of chronic noise exposure on the microbiome-gut-brain axis in senescence-accelerated prone mice: implications for Alzheimer's disease.

BACKGROUND: Chronic noise exposure is associated with neuroinflammation and gut microbiota dysregulation and increases the risk of Alzheimer's disease (AD). Environmental hazards are also thought to be associated with genetic susceptibility factors that increase AD pathogenesis. However, there is limited experimental evidence regarding the link between chronic noise stress and microbiome-gut-brain axis alterations, which may be closely related to AD development. METHODS: The aim of the present study was to systematically investigate the effects of chronic noise exposure on the microbiome-gut-brain axis in the senescence-accelerated mouse prone 8 (SAMP8) strain. We established SAMP8 mouse models to examine the consequences of noise exposure on the microbiome-gut-brain axis. Hippocampal amyloid-ß (Aß) assessment and the Morris water maze were used to evaluate AD-like changes, 16S ribosomal RNA sequencing analyses were used for intestinal flora measurements, and assessment of endothelial tight junctions and serum neurotransmitter and inflammatory mediator levels, as well as fecal microbiota transplant, was conducted to explore the underlying pathological mechanisms. RESULTS: Chronic noise exposure led to cognitive impairment and Aß accumulation in young SAMP8 mice, similar to that observed in aging SAMP8 mice. Noise exposure was also associated with decreased gut microbiota diversity and compositional alterations. Axis-series studies showed that endothelial tight junction proteins were decreased in both the intestine and brain, whereas serum neurotransmitter and inflammatory mediator levels were elevated in young SAMP8 mice exposed to chronic noise, similar to the observations made in the aging group. The importance of intestinal bacteria in noise exposure-induced epithelial integrity impairment and Aß accumulation was further confirmed through microbiota transplantation experiments. Moreover, the effects of chronic noise were generally intensity-dependent. CONCLUSION: Chronic noise exposure altered the gut microbiota, accelerated age-related neurochemical and inflammatory dysregulation, and facilitated AD-like changes in the brain of SAMP8 mice.

Effects of the differential expression of ZO-1 and ZO-2 on podocyte structure and function.

Glomerular podocytes in the kidney originate from columnar epithelial cells possessing tight junctions. During podocyte differentiation, tight junctions are replaced by slit diaphragms, which are formed between foot processes and function as a blood filtration barrier. Although the expression of most tight junction components is suppressed during podocyte differentiation, several components, including ZO-1 and ZO-2, are consistently expressed. We recently showed that podocyte-specific deletion of ZO-1 gene impaired slit diaphragm formation, leading to proteinuria and glomerular sclerosis. Here, we address the relevance of ZO-2, whose sequence is highly similar to ZO-1, in the maintenance of the structure and function of podocytes. In glomerular development, the spatiotemporal expression of ZO-2 was similar to that of ZO-1 until the capillary loop stage. Subsequently, the distribution patterns of ZO-1 and ZO-2 diverged at the maturation stage, when slit diaphragms are formed. This divergence could partly rely on the ability of ZO-2 to interact with the slit diaphragm membrane proteins. Podocyte-specific deletion of the ZO-2 gene did not cause overt defects; however, double knockout of ZO-1 and ZO-2 genes accelerated the defects observed in ZO-1 knockout mice. These results suggest that ZO-2 plays supportive roles in the ZO-1-dependent regulation of podocyte filtration barrier.

Cryptogenic cholestasis in young and adults: ATP8B1, ABCB11, ABCB4, and TJP2 gene variants analysis by high-throughput sequencing.

BACKGROUND: Mutations in ATP-transporters ATPB81, ABCB11, and ABCB4 are responsible for progressive familial intrahepatic cholestasis (PFIC) 1, 2 and 3, and recently the gene for tight junction protein-2 (TJP2) has been linked to PFIC4. AIM: As these four genes have been poorly studied in young people and adults, we investigated them in this context here. METHODS: In patients with cryptogenic cholestasis, we analyzed the presence of mutations by high-throughput sequencing. Bioinformatics analyses were performed for mechanistic and functional predictions of their consequences on biomolecular interaction interfaces. RESULTS: Of 108 patients, 48 whose cause of cholestasis was not established were submitted to molecular analysis. Pathogenic/likely pathogenic mutations were found in ten (21%) probands for 13 mutations: two in ATP8B 1, six in ABCB11, two in ABCB4, three in TJP2. We also identified seven variants of uncertain significance: two in ATP8B1, one in ABCB11, two in ABCB4 and two in TJP2. Finally, we identified 11 benign/likely benign variants. Patients with pathogenic/likely pathogenic mutations had higher levels of liver stiffness (measured by FibroScan®) and bile acids, as well as higher rates of cholestatic histological features, compared to the patients without at least likely pathogenic mutations. The multivariate analysis showed that itching was the only independent factor associated with disease-causing mutations (OR 5.801, 95% CI 1.244-27.060, p = 0.025). CONCLUSIONS: Mutations in the genes responsible for PFIC may be involved in both young and adults with cryptogenic cholestasis in a considerable number of cases, including in heterozygous status. Diagnosis should always be suspected, particularly in the presence of itching.