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Periostin Signaling Pathway
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Hypothalamic Expression of Galanin Is Dysregulated During Cholestatic Liver Injury and Contributes to Hyperplastic Cholangiocyte Proliferation Matthew McMillin, Gabriel A. Frampton, Cheryl Galindo, Sharon DeMorrow
Cholesterol Augments High Fat Diet in Accelerating Liver Carcinogenesis: Roles of NASH, Oxidative Stress, DNA Damage and Hepatocyte Proliferation Lixia Xu, Sharon Pok, Evi Arfianti, Francis K. L. Chan, Jun Yu, Narci C. Teoh, Geoff C Farrell
During the course of cholestatic liver diseases, cholangiocytes exhibit marked proliferative capacity followed by cholangiocyte loss. A hallmark feature of cholestasis is increased concentrations of serum bile acids. We have previously shown alteration of hypothalamic-derived hormones during cholestatic liver injury as a result of aberrant bile acid signaling in the hypothalamus via an ASBT/FXR-dependent mechanism. This dysregulated signaling may regulate the proliferative response of cholangiocytes in a paracrine manner. Galanin is a hypothalamic neuropeptide that controls the growth hormone releasing hormone (GHRH)/ growth hormone (GH) axis. AIMS: The aims of this study were to i) assess the expression of Galanin in the hypothalamus during cholestatic liver injury, ii) determine the effects of bile acid signaling on hypothalamic expression of Galanin and iii) evaluate the effects of central activation of Galanin signaling on cholangiocyte proliferation. Methods: Male Sprague-Dawley rats underwent sham or BDL surgery. In parallel, central administration of the bile acids cholic acid (CA), deoxycholic acid (DCA) and taurocholic acid (TCA; all at 10 pmol icv) were performed and tissue was collected after 3 hr. The expression of Galanin and GHRH in the hypothalamus and GH in the pituitary was assessed by real time PCR and immnunofluorescence. In vitro, hypothalamic neurons (m-hypo-A) were treated with the above-mentioned bile acids as well as the FXR agonist fexaramine (10 and 100 nM) for 4 hr, and galanin expression was assessed. Lastly, a synthetic Galanin analogue (M617) was infused into the brain (100 pmol/day icv) of sham and BDL rats; intrahepatic bile duct mass (IBDM) was assessed by real time PCR and immunohistochemistry using cytokeratin 19 as a cholangiocyte marker. Results: Hypothalamic Galanin expression was upregulated 1 to 7 days after BDL surgery, paralleled by an increase in GHRH and GH expression. Treatment of hypothalamic neurons with DCA, CA, or TCA increased Galanin mRNA expression in vitro and in vivo, with DCA having the greatest effect. Furthermore, treatment of m-hypoA cells with fexaramine increased Galanin expression to a similar degree. Lastly, central administration of M617 increased the expression of GHRH and GH in the hypothalamus and pituitary, respectively, and increased CK-19 mRNA expression and IBDM in sham and BDL animals. Conclusions: Our data provide further evidence for dysregulation of hypothalamic peptides during cholestatic liver injury. Specifically, the hypothalamic expression of Galanin and subsequent activation of the GHRH/GH axis is upregulated via a mechanism involving bile acid signaling. This Galanin/GHRH/GH axis may contribute to cholangiocyte proliferation during cholestatic liver injury and may be an effective therapeutic target for the maintenance of biliary mass during cholestatic liver diseases.
Background & Aims: Obesity, dietary cholesterol and NAFLD are risk factors for hepatocellular carcinoma (HCC). Dietary and genetic obesity accelerate experimental hepatocarcinogenesis, but it is unclear which nutrients play an independent role. We investigated the effects of sugar, saturated fat and cholesterol intake on hepatocarcinogenesis, exploring the role of associated liver disease, oxidant stress, DNA damage and tumor-promoting pathways. Materials & Methods: Male C57BI/6J mice injected 5 mg/kg diethylnitrosamine (DEN) intraperitoneally (i.p.) at day 12-15 (saline for controls) were fed 6-32 weeks of age one of four diets: normal chow (NC), high sucrose (HS), high saturated-fat (HSF), or combined high saturated-fat, high sucrose and cholesterol (Atherogenic, Ath). I.p. glucose tolerance test (IPGTT) was performed 2 weeks prior to sacrifice. Results: 90-100% of DEN-treated mice fed a HS, HSF and Ath diet developed HCC, compared with 67% NC-fed mice. In Ath-fed mice, HCCs were larger, more numerous and 40% developed lung metastases. Body weight of mice fed all obesigenic diets was similarly increased compared to NC, but liverto-body weight was significantly higher in Ath-fed mice. Irrespective of DEN treatment, all diet-fed mice (vs NC) developed hyperglycemia and were similarly insulin resistant. Compared with NC and other dietary groups, serum ALT and cholesterol were significantly raised in Ath-fed mice. Non-tumorous livers of Ath-fed mice showed NASH, whereas HSFfed and HS-fed mice showed only steatosis. Consistent with presence of NASH, only Athfed mice exhibited hepatic oxidant stress evident by increased expression of Nrf1, a redox transcription factor. Ataxia-telangiectasia-mutated (ATM) kinase, a sensor and regulator of DNA damage response, was up-regulated in livers from mice fed modified diets, with downregulation of the cell cycle regulatory protein pChk2 compared with NC. Proliferative markers, PCNA and cyclin D1 were enhanced in both HCCs and surrounding liver only in Ath-fed mice; cyclin E and cdk2 increased in livers from both Ath and HSF-fed mice. p53 and Rb protein were also markedly enhanced in HCCs from Ath and HSF-fed mice, but expression of p21, the downstream cell cycle regulator of p53, remained unaltered. Conclusion: High dietary cholesterol intake with saturated fat accelerates hepatocarcinogenesis to a greater extent than high saturated-fat or high sucrose alone, and this is associated with development of NASH. In addition to obesity-related hyperinsulinemia and hyperglycemia found with all diets, cholesterol-related NASH induced oxidative stress and DNA damage. Associated acceleration of proliferative drive in hepatocytes could provide the growth pressure that promotes HCC progression. This dietary NASH model should prove useful to study the mechanisms by which NAFLD and obesity leads to HCC.
767 769 Inhibition of FGF19 Signaling Decreases Proliferation and Sensitizes Cholangiocytes to Apoptosis Ashley M. Mohr, Mary A. Smith, Sathish Kumar Natarajan, Cody Wehrkamp, Justin L. Mott
Vitamin D As a Chemopreventive Agent for Hepatocellular Carcinoma in the Context of Impaired TGF-β Signaling Pathway Lior H. Katz, Andrea Cortes, Vivek Shukla, Keigo Machida, Hidekazu Tsukamoto, Kirti Shetty, Aiwu R. He, Lynt B. Johnson, Jian Chen, Ju-Seog Lee, Randa El-Zein, Lopa Mishra
Expression and release of growth factors and hormones is a mechanism employed by hepatobiliary cells to increase proliferation and evade apoptosis. Fibroblast growth factor 19 (FGF19), a secreted hormone of the digestive system, binds its cognate receptor tyrosine kinase, FGFR4 (fibroblast growth factor receptor 4), to mediate cell survival and proliferation. We identified secreted FGF19 and FGFR4 expression in an immortalized cholangiocyte cell line, H69. Inhibition of FGFR signaling (using a small molecule inhibitor, PD173074) decreased proliferation in H69 cells at 24, 48 and 72 hours. Due to this effect, we decided to investigate the FGF19/FGFR4 signaling pathway in cholangiocarcinoma (CCA). FGFR4 is overexpressed in many cancers and the signaling pathway is amplified leading to increased proliferation and resistance to chemotherapy. FGF19/FGFR4 signaling has been identified in hepatocellular carcinoma, but has not yet been evaluated in CCA. Our studies have identified tumor cell expression of both the FGF19 ligand and the transmembrane receptor FGFR4 in CCA cell lines, KMCH and Mz-ChA-1. These cell lines (KMCH and Mz-ChA-1) in addition to H69 cells secrete FGF19 both in normal growth medium and in serum-free conditions. To examine pathway activation, we stimulated KMCH cells with recombinant FGF19, which lead to an increase in phosphorylated ERK, while treatment with the FGFR4 inhibitor decreased levels of phosphorylated ERK. In addition, treatment with the FGFR inhibitor sensitized KMCH and Mz-ChA-1 cells to TRAIL-induced apoptosis, measured by nuclear morphology or biochemically by caspase 3/7 activity. Treatment of KMCH and MzChA-1 cells with PD173074 was sufficient to significantly decrease proliferation at 48, and 72 hours. To begin to evaluate mechanistic pathways involved, we examined levels of FGFR4, the S-phase protein MCM7 (minichromosome maintenance complex component 7) and miR-93 (a microRNA encoded in an intron of MCM7) in a rat model of cholangiocarcinoma. FGFR4 was highly expressed in the tumor. MCM7 and miR-93 were both overexpressed in tumors compared to normal liver. To determine if the FGFR pathway is involved in miR93 regulation, we examined cellular microRNA levels after treatment of CCA cells with PD173074. FGFR inhibition resulted in a significant decrease in miR-93 levels. Furthermore, we found that levels of XIAP (X-linked inhibitor of apoptosis) were lower upon FGFR inhibition, providing a mechanistic target that FGF19/FGFR4 can utilize to increase survival. We conclude that cholangiocytes secrete FGF19 and activate a cell autonomous signaling pathway that reduces apoptosis and promotes proliferation in both normal cholangiocytes and malignant cell lines. The regulatory mediators by which FGF19/FGFR4 signaling regulates MCM7, miR-93, and XIAP, are currently being evaluated.
Hepatocellular carcinoma (HCC) is the third most common cause of cancer related death worldwide. Vitamin D (VD) has been implicated in the prevention of multiple cancers- some with inactivation of TGF-β signaling. Recently, inactivation of TGF- β signaling has been associated with HCC. We analyzed whole genome data for TGF- βsignaling and examined the role of VD in the context of TGF- β inactivation in HCC. Methods: Databases of HCC Genomics (COSMIC) and transcriptomics (TCGA) were analyzed. The effect of calcitriol on cell proliferation was measured in HCC cell lines; Hep-G2 and MEF cells, with knocked down TGF-β/β2-spectrin (β2SP). Wild-type (WT), Sptbn1+/- and Smad3+/- mice were fed with diets containing 200 IU VD/kg or 10,000 IU VD/kg for 9 weeks. Hepatocyte proliferation was analyzed through Ki67 staining. The expression of cyclin D1 was evaluated by Western blotting. A whole genome gene expression profiling array was performed from liver samples of those mice using Illumina MouseWG-6 v2.0 gene expression arrays®. Lastly, liver samples from patients with HCC who had received VD supplementation were evaluated by immunohistochemistry. Results: Whole genome data revealed aberrant TGF- β signaling in ~70% of HCCs. Treatment with calcitriol suppressed the growth of HCC cell lines, regardless of their TGF-β pathway status. Inhibition of proliferation was noted in WT MEFs as well as in the Sptbn1+/- and Sptbn1-/- cells. High dose VD reduced hepatic proliferation and cyclin D1 levels in Sptbn1+/- and Smad3+/- mutants, and in WT mice. The microarray data demonstrated the most significant changes in gene expression were associated with acute phase inflammatory response (p<0.005). Validation studies showed a significant fold change difference of P-PARA, OAZ1, TLR7 and TLR 9 after high dose VD diet between WT mice and the mutants (p<0.05). VD supplementation to patients with HCC was associated with higher expression of β2SP (p<.0001) and TβRII (p=.013) compared with that of patients who did not receive the supplement. Conclusions: Whole genome data revealed aberrant TGF- β signaling in ~ 70% of HCCs. VD suppresses the proliferation of HCC cell lines as well as normal hepatocytes potentially through restoring of TGF- β tumor suppressor signaling. VD modulates inflammation markedly differently in the setting of TGF- βsignaling inactivation. Taken together, these results suggest that VD treatment strategies could potentially be pivotal in prevention and treatment of HCC. 770 Sulfatase2 (SULF2) Promotes Angiogenesis in Hepatocellular Carcinoma Partly Through the TGFβ1/Periostin Signaling Pathway Gang Chen, Alexander Miamen, Renumathy Dhanasekaran, Catherine D. Moser, Shaoshan Han, Yichun Chen, Yong Fang, Dongye Yang, Hassan M. Shaleh, Chunling Hu, Snorri Thorgeirsson, Lewis R. Roberts Background/Aim: The growth of hepatocellular carcinoma (HCC) requires the formation of new blood vessels, which has provided a strong rationale for antiangiogenic strategies for therapy of HCC. However, the molecular pathways involved in HCC angiogenesis are
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AASLD Abstracts
AASLD Abstracts
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AASLD Abstracts
incompletely characterized. The heparin-degrading endosulfatase 2, SULF2, affects the activity of a number of heparin-binding growth factors by removing 6-O-sulfates from heparan sulfate proteoglycans (HSPGs). In our previous study, we showed that SULF2 can increase HCC growth and that high SULF2 expression predicts a worse prognosis after resection. HSPGs are known to mediate angiogenesis. Therefore, we investigated whether SULF2 can promote angiogenesis of HCC and explored its mechanism. Methods: The correlation between SULF2 mRNA and angiogenesis associated gene mRNAs was assessed in 139 resected HCCs by cDNA microarray analysis. Angiogenesis associated gene expression and microvascular density were assessed in diethyl nitrosamine (DEN)-induced HCC tumors in SULF2 transgenic mice as well as in xenografts established from SULF2-transfected Hep3B HCC cells. Cell viability, cell migration and tube formation ability of human hepatic sinusoidal endothelial cells (HHSEC) and human umbilical vein endothelial cells (HUVEC) were assessed after co-culture with SULF2-negative Hep3B cells transfected with SULF2 or SULF2-expressing Huh7 cells transfected with shRNA targeting SULF2. Angiogenic signaling pathways were assayed by Western blotting. Results: SULF2 mRNA expression was positively correlated with key angiogenesis associated genes which include CD31, VACAM1, VEGF B, and SDF1 in 139 resected HCCs. Microvascular density was increased in tumor tissues from both SULF2 up-regulated mouse models. Upregulation of SULF2 in Hep3B cells increased cell viability, cell migration and tube formation ability of HHSEC and HUVEC in a paracrine fashion; conversely knockdown of SULF2 in Huh7 cells reversed these morphologic features. Assessment of angiogenic pathways showed that the periostin pathway was activated in HHSEC cells after co-culture with SULF2 up-regulated Hep3B cells. The downstream factors p-AKT and p-FAK were also increased. The changes in p-AKT and p-FAK were further enhanced by activation of the TGF- β1 signaling pathway. The same association of TGF- β1, periostin, and p-FAK signaling changes was observed in both SULF2 up-regulated mouse HCC tumor models. Conclusions: Together, these findings suggest that SULF2 promotes angiogenesis of HCCs by increasing the angiogenic potency of HHSEC in a paracrine fashion. The TGFβ1/periostin signaling pathway plays an important role in regulating SULF2mediated angiogenesis of HCC. The SULF2/TGF β1/periostin pathway may be a rational target for anti-angiogenic therapy of HCC.
PLINK was used for statistical analysis. Results: Sequencing of ABCB4 identified 1 splice site, 9 Synonymous and 9 NS variants. Of note, one NS variant, p.A934T, described in LPAC patients, was present in 4 subjects in the ET group and none in the SNL group. All 4 affected individuals were of African American (AA) race (20 AA in the ET and 13 AA in the SNL group). Population analyses indicate that p.A934T is differentially represented in US racial subgroups, with an AA heterozygote frequency of 2.45%, compared with <0.01% among European Americans. Armitage trend test demonstrated p.A934T is significantly associated with ET outcome when examining all races (p=0.04). Other variants reported in LPAC or PFIC3 patients (p.T34M, p.T175A, p.R590Q, p.R652G, p.R788Q, p.E1058K) were similarly distributed among ET and SNL groups. Conclusions: This is the first WES study to discover genetic determinants of clinical outcomes in BA and identified ABCB4 as a biologically plausible candidate gene modifier. Heterozygosity for p.A934T is overrepresented among AA subjects with BA requiring early transplantation post-HPE. This finding underscores the utility of WES and subsequent validation studies of one candidate gene in a large, well-phenotyped cohort of racially diverse subjects with BA. With further application, this approach is poised to discover additional genetic contributors to BA outcomes. Support: NIH (TL1TR000456, U01DK062456, U01DK062470) & the Bauer, Spain & Alpard Foundations 846 Mutations in Tight Junction Protein 2 Underlie a Spectrum of Cholestatic Liver Disease Melissa Sambrotta, Sandra Strautnieks, Laura Brett, Suzanne Davison, Richard J. Thompson Background. Tight Junction Protein 2 is a cytoplasmic protein essential for the assembly of cell-cell junctional structures. It is encoded by TJP2. In 2003 a single, incompletely penetrant, missense mutation was identified in TJP2 in Amish patients with hypercholanaemia, in the absence of significant liver disease. We have recently shown that homozygous protein-truncating mutations in TJP2 are present in patients with severe early-onset cholestatic liver disease. Most of these patients required liver transplantation. Aim and Methods. In order to explore the wider phenotypic spectrum associated with mutations in TJP2, a cohort of 53 patients with severe early-onset of normal GGT cholestasis was selected. No mutations in the known progressive familial intrahepatic cholestasis genes ( ABCB11 and ATP8B1) had been identified. Genetic testing was undertaken using a combination of next-generation sequencing technology and Sanger DNA sequencing. Pathogenicity of missense mutations was determined with in silico analysis. Results. Genetic analysis revealed homozygous mutations in 8 patients. Three deletions and 2 nonsense mutation were identified, all predicted to lead to the truncation of the protein. In three families missense mutations were found. p.Gly737Arg was present in one patient who required liver transplantation at 13 months. However p.His788Leu was found in 2 unrelated families. Both patients had severe earlyonset cholestasis, which subsequently remitted. One of these patients relapsed at 5 years, but remitted again at age 7 years. Conclusion. We have previously described 8 families, including 12 individuals, with 8 different protein truncating mutations. We now report a further 8 families. Six manifest persistent cholestasis: five had protein-truncating mutations, and one had a homozygous missense mutation. The other 2 individuals had remitting cholestasis and were both homozygous for the same missense mutation. TJP2 was previously shown to underlie a condition with minimal liver disease. In addition to our recent identification of mutations in patients with progressive familial intrahepatic cholestasis, we now show that it can also lead to an intermediate phenotype. It is therefore clear that TJP2 deficiency represents a spectrum of liver disease phenotypes.
771 Mitochondria-Targeting Antioxidant Attenuates Cholestasis-Induced Liver Injury in Mice Yanjun Shi, Hasibur Rehman, Yasodha Krishnasamy, Venkat K. Ramshesh, Rick G. Schnellmann, John J. Lemasters, Michael P. Murphy, Zhi Zhong Background: Our previous study showed that the mitochondrial permeability transition (MPT) occurs in the liver after cholestasis, leading to cell death. Aim: Because reactive oxygen species (ROS) are a known stimulator of the MPT, we investigated whether MitoQ, an orally active antioxidant that accumulates in mitochondria, attenuates cholestatic liver injury caused by bile duct ligation (BDL) in mice. Methods: MitoQ (500 μM) was added to drinking water. Three days after starting MitoQ feeding, mice were subjected to BDL, and livers were harvested 1 and 3 days later. Mitochondrial depolarization and cell death were detected in living mice by intravital confocal/multiphon microscopy of rhodamine 123 (Rh123) and propidium iodide (PI), respectively, and onset of the MPT was assessed by entry of calcein into mitochondria. Results: 4-Hydroxynonenal adducts (4-HNE) increased markedly after BDL, indicating ROS formation, and MitoQ blunted increases in 4-HNE. Rh123 fluorescence was punctate in virtually all hepatocytes in control mice, indicating mitochondrial polarization. At 6 h after BDL, Rh123 fluorescence disappeared in many hepatocytes (~18/hpf) whereas PI-positive non-viable hepatocytes were <1/hpf, indicating mitochondrial depolarization prior to cell death. Calcein resided in the cytosol in hepatocytes of sham-operated mice but entered mitochondria 6 h after BDL, confirming the onset of the MPT in vivo. MitoQ decreased the number of depolarized hepatocytes after BDL by 72%. Serum ALT increased from 46 U/L before surgery to 3300 and 1700 U/L at 1 and 3 days, respectively, after BDL. ALT was blunted by 85% 1 day after BDL in mice given MitoQ. Focal necrosis occurred in 17% of liver tissue 1 day after BDL, and MitoQ attenuated necrosis to 5%. TUNEL-positive cells were barely detectable in livers after sham operation but increased to 4% and 2% at 1 and 3 days, respectively, after BDL and MitoQ decreased TUNEL-positive cells to 0.5%. Cleaved caspase-3 was barely detectable in livers from shamoperated mice, increased markedly after BDL, and was decreased by MitoQ. Conclusion. Taken together, we suggest that MitoQ decreases liver injury after BDL by blocking ROSinduced MPT onset. This mitochondria-targeting antioxidant may be a promising therapy to prevent early stage cholestatic liver injury (NIDDK).
847 The Types of Inflammatory Bowel Disease (IBD) Predispose to Distinct Clinical Phenotypes of Primary Sclerosing Cholangitis (PSC) in Children Keaton R. Jones, Philip Bufler, Alexander G. Miethke Background: PSC is a chronic progressive cholangiopathy which is commonly associated with IBD. Since the gut-liver axis is considered to play an important role in the pathogenesis of PSC we hypothesized that the type of IBD determined the disease course of pediatric onset PSC. Methods: In this retrospective study, medical records of 52 patients who received medical care for PSC at a single institution between 2009 and 2013 were reviewed. Results: The clinical diagnosis of PSC was confirmed in all 52 subjects based on review of reports of hepatobiliary imaging and liver biopsy. A diagnosis of IBD was established by endoscopy in 43 (83%), comprising of Crohn disease (CD) in 16 (31%) and ulcerative colitis (UC) in 27 (52%) patients. Ethnicity was primarily Caucasian in both groups (81 vs 77% in CD vs UC), but males predominated in the CD group (62 vs 46% males in CD vs UC). Mean age of diagnosis of PSC was 10.8 years in CD-PSC and 12.0 in UC-PSC patients. CD and PSC were often diagnosed simultaneously whereas the diagnosis of UC preceded detection of abnormal liver enzymes by a mean of 7 months. Maintenance therapy of colitis differed between both groups in regards to use of immunomodulator and corticosteroid therapy. Infliximab was used in 43% of CD- and 27 % of UC-PSC subjects. 9 patients (7 with PSCUC) with end-stage liver disease were referred for liver transplantation and were excluded from subsequent analysis. Of 34 patients with PSC, IBD and native liver, 6 (18%) were found to have autoimmune sclerosing cholangitis based on positive ANA serology and liver histomorphology with interface hepatitis (5 in UC- group). For 18 subjects with laboratory values available at the time of diagnosis of PSC, serum GGT levels and white blood cell counts (WBC) were significantly higher in CD- than in UC patients (see table). During follow up, peak serum total bilirubin levels and those obtained at the most recent visit were higher in CD- PSC patients. Importantly, elevated levels of biomarkers of cholestasis were associated with a higher prevalence of extrahepatic biliary disease in CD-PSC patients. Narrowing or dilatation with caliber change affecting the common hepatic or common bile ducts were detected by ERCP or MRCP in 9/12 PSC-CD (75%) compared with 7/ 21 PSCUC patients (33%; p=0.03). Conclusion: Compared with UC, concomitant CD predisposes in pediatric onset PSC to extrahepatic bile duct injury and greater degree of cholestasis, as gauged by serum biochemistries. How disease pathogenesis and treatment of colitis are linked to short and long-term outcomes in CD- and UC-PSC patients, especially progression of bile duct epithelial injury and biliary fibrosis, requires further investigation.
845 Whole Exome Sequencing Identifies ABCB4 Gene Variants As Modifiers of Biliary Atresia Outcomes Anya Mezina, Khanjan Gandhi, Aniko Sabo, Donna Muzny, Richard Gibbs, Madhuri Hegde, Saul J. Karpen Background: The role of genetic factors in determining clinical outcomes in biliary atresia (BA) after Kasai hepatoportoenterostomy (HPE) are unknown. To begin to address this, we performed a pilot study of whole exome sequencing (WES) in 20 BA patients from the NIDDK-supported ChiLDREN dataset to identify variants in genes that stratify between two disparate outcome groups: 10 who underwent early transplant prior to age 2 (ET) compared to 10 who survived beyond age 4 with native liver (SNL) and normal platelet count (>150,000). WES identified ~ 50 candidate genes with disproportionately more non-synonymous (NS) variants in the ET compared to SNL group. Among this list was the ABCB4 phospholipid floppase gene, which, if verified, would provide a pathophysiological link to worsening cholangiopathy in BA since ABCB4 gene mutations are implicated in liver diseases such as Low Phospholipid Associated Cholelithiasis (LPAC) and Progressive Familial Intrahepatic Cholestasis 3 (PFIC3). We hypothesized that deleterious variants in ABCB4 would modulate phenotypic severity and sought to validate this in a larger cohort of ET and SNL ChiLDREN subjects. Methods: Sanger DNA sequencing was performed for all 27 coding exons and the promoter region of the ABCB4 gene from 195 ChiLDREN subjects stratified as SNL (n=97) or ET (n=98). Variants were called by comparison to reference NM_018849.2.
AASLD Abstracts
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Hypothalamic Expression of Galanin Is Dysregulated During Cholestatic Liver Injury and Contributes to Hyperplastic Cholangiocyte Proliferation Matthew McMillin, Gabriel A. Frampton, Cheryl Galindo, Sharon DeMorrow
Cholesterol Augments High Fat Diet in Accelerating Liver Carcinogenesis: Roles of NASH, Oxidative Stress, DNA Damage and Hepatocyte Proliferation Lixia Xu, Sharon Pok, Evi Arfianti, Francis K. L. Chan, Jun Yu, Narci C. Teoh, Geoff C Farrell
During the course of cholestatic liver diseases, cholangiocytes exhibit marked proliferative capacity followed by cholangiocyte loss. A hallmark feature of cholestasis is increased concentrations of serum bile acids. We have previously shown alteration of hypothalamic-derived hormones during cholestatic liver injury as a result of aberrant bile acid signaling in the hypothalamus via an ASBT/FXR-dependent mechanism. This dysregulated signaling may regulate the proliferative response of cholangiocytes in a paracrine manner. Galanin is a hypothalamic neuropeptide that controls the growth hormone releasing hormone (GHRH)/ growth hormone (GH) axis. AIMS: The aims of this study were to i) assess the expression of Galanin in the hypothalamus during cholestatic liver injury, ii) determine the effects of bile acid signaling on hypothalamic expression of Galanin and iii) evaluate the effects of central activation of Galanin signaling on cholangiocyte proliferation. Methods: Male Sprague-Dawley rats underwent sham or BDL surgery. In parallel, central administration of the bile acids cholic acid (CA), deoxycholic acid (DCA) and taurocholic acid (TCA; all at 10 pmol icv) were performed and tissue was collected after 3 hr. The expression of Galanin and GHRH in the hypothalamus and GH in the pituitary was assessed by real time PCR and immnunofluorescence. In vitro, hypothalamic neurons (m-hypo-A) were treated with the above-mentioned bile acids as well as the FXR agonist fexaramine (10 and 100 nM) for 4 hr, and galanin expression was assessed. Lastly, a synthetic Galanin analogue (M617) was infused into the brain (100 pmol/day icv) of sham and BDL rats; intrahepatic bile duct mass (IBDM) was assessed by real time PCR and immunohistochemistry using cytokeratin 19 as a cholangiocyte marker. Results: Hypothalamic Galanin expression was upregulated 1 to 7 days after BDL surgery, paralleled by an increase in GHRH and GH expression. Treatment of hypothalamic neurons with DCA, CA, or TCA increased Galanin mRNA expression in vitro and in vivo, with DCA having the greatest effect. Furthermore, treatment of m-hypoA cells with fexaramine increased Galanin expression to a similar degree. Lastly, central administration of M617 increased the expression of GHRH and GH in the hypothalamus and pituitary, respectively, and increased CK-19 mRNA expression and IBDM in sham and BDL animals. Conclusions: Our data provide further evidence for dysregulation of hypothalamic peptides during cholestatic liver injury. Specifically, the hypothalamic expression of Galanin and subsequent activation of the GHRH/GH axis is upregulated via a mechanism involving bile acid signaling. This Galanin/GHRH/GH axis may contribute to cholangiocyte proliferation during cholestatic liver injury and may be an effective therapeutic target for the maintenance of biliary mass during cholestatic liver diseases.
Background & Aims: Obesity, dietary cholesterol and NAFLD are risk factors for hepatocellular carcinoma (HCC). Dietary and genetic obesity accelerate experimental hepatocarcinogenesis, but it is unclear which nutrients play an independent role. We investigated the effects of sugar, saturated fat and cholesterol intake on hepatocarcinogenesis, exploring the role of associated liver disease, oxidant stress, DNA damage and tumor-promoting pathways. Materials & Methods: Male C57BI/6J mice injected 5 mg/kg diethylnitrosamine (DEN) intraperitoneally (i.p.) at day 12-15 (saline for controls) were fed 6-32 weeks of age one of four diets: normal chow (NC), high sucrose (HS), high saturated-fat (HSF), or combined high saturated-fat, high sucrose and cholesterol (Atherogenic, Ath). I.p. glucose tolerance test (IPGTT) was performed 2 weeks prior to sacrifice. Results: 90-100% of DEN-treated mice fed a HS, HSF and Ath diet developed HCC, compared with 67% NC-fed mice. In Ath-fed mice, HCCs were larger, more numerous and 40% developed lung metastases. Body weight of mice fed all obesigenic diets was similarly increased compared to NC, but liverto-body weight was significantly higher in Ath-fed mice. Irrespective of DEN treatment, all diet-fed mice (vs NC) developed hyperglycemia and were similarly insulin resistant. Compared with NC and other dietary groups, serum ALT and cholesterol were significantly raised in Ath-fed mice. Non-tumorous livers of Ath-fed mice showed NASH, whereas HSFfed and HS-fed mice showed only steatosis. Consistent with presence of NASH, only Athfed mice exhibited hepatic oxidant stress evident by increased expression of Nrf1, a redox transcription factor. Ataxia-telangiectasia-mutated (ATM) kinase, a sensor and regulator of DNA damage response, was up-regulated in livers from mice fed modified diets, with downregulation of the cell cycle regulatory protein pChk2 compared with NC. Proliferative markers, PCNA and cyclin D1 were enhanced in both HCCs and surrounding liver only in Ath-fed mice; cyclin E and cdk2 increased in livers from both Ath and HSF-fed mice. p53 and Rb protein were also markedly enhanced in HCCs from Ath and HSF-fed mice, but expression of p21, the downstream cell cycle regulator of p53, remained unaltered. Conclusion: High dietary cholesterol intake with saturated fat accelerates hepatocarcinogenesis to a greater extent than high saturated-fat or high sucrose alone, and this is associated with development of NASH. In addition to obesity-related hyperinsulinemia and hyperglycemia found with all diets, cholesterol-related NASH induced oxidative stress and DNA damage. Associated acceleration of proliferative drive in hepatocytes could provide the growth pressure that promotes HCC progression. This dietary NASH model should prove useful to study the mechanisms by which NAFLD and obesity leads to HCC.
767 769 Inhibition of FGF19 Signaling Decreases Proliferation and Sensitizes Cholangiocytes to Apoptosis Ashley M. Mohr, Mary A. Smith, Sathish Kumar Natarajan, Cody Wehrkamp, Justin L. Mott
Vitamin D As a Chemopreventive Agent for Hepatocellular Carcinoma in the Context of Impaired TGF-β Signaling Pathway Lior H. Katz, Andrea Cortes, Vivek Shukla, Keigo Machida, Hidekazu Tsukamoto, Kirti Shetty, Aiwu R. He, Lynt B. Johnson, Jian Chen, Ju-Seog Lee, Randa El-Zein, Lopa Mishra
Expression and release of growth factors and hormones is a mechanism employed by hepatobiliary cells to increase proliferation and evade apoptosis. Fibroblast growth factor 19 (FGF19), a secreted hormone of the digestive system, binds its cognate receptor tyrosine kinase, FGFR4 (fibroblast growth factor receptor 4), to mediate cell survival and proliferation. We identified secreted FGF19 and FGFR4 expression in an immortalized cholangiocyte cell line, H69. Inhibition of FGFR signaling (using a small molecule inhibitor, PD173074) decreased proliferation in H69 cells at 24, 48 and 72 hours. Due to this effect, we decided to investigate the FGF19/FGFR4 signaling pathway in cholangiocarcinoma (CCA). FGFR4 is overexpressed in many cancers and the signaling pathway is amplified leading to increased proliferation and resistance to chemotherapy. FGF19/FGFR4 signaling has been identified in hepatocellular carcinoma, but has not yet been evaluated in CCA. Our studies have identified tumor cell expression of both the FGF19 ligand and the transmembrane receptor FGFR4 in CCA cell lines, KMCH and Mz-ChA-1. These cell lines (KMCH and Mz-ChA-1) in addition to H69 cells secrete FGF19 both in normal growth medium and in serum-free conditions. To examine pathway activation, we stimulated KMCH cells with recombinant FGF19, which lead to an increase in phosphorylated ERK, while treatment with the FGFR4 inhibitor decreased levels of phosphorylated ERK. In addition, treatment with the FGFR inhibitor sensitized KMCH and Mz-ChA-1 cells to TRAIL-induced apoptosis, measured by nuclear morphology or biochemically by caspase 3/7 activity. Treatment of KMCH and MzChA-1 cells with PD173074 was sufficient to significantly decrease proliferation at 48, and 72 hours. To begin to evaluate mechanistic pathways involved, we examined levels of FGFR4, the S-phase protein MCM7 (minichromosome maintenance complex component 7) and miR-93 (a microRNA encoded in an intron of MCM7) in a rat model of cholangiocarcinoma. FGFR4 was highly expressed in the tumor. MCM7 and miR-93 were both overexpressed in tumors compared to normal liver. To determine if the FGFR pathway is involved in miR93 regulation, we examined cellular microRNA levels after treatment of CCA cells with PD173074. FGFR inhibition resulted in a significant decrease in miR-93 levels. Furthermore, we found that levels of XIAP (X-linked inhibitor of apoptosis) were lower upon FGFR inhibition, providing a mechanistic target that FGF19/FGFR4 can utilize to increase survival. We conclude that cholangiocytes secrete FGF19 and activate a cell autonomous signaling pathway that reduces apoptosis and promotes proliferation in both normal cholangiocytes and malignant cell lines. The regulatory mediators by which FGF19/FGFR4 signaling regulates MCM7, miR-93, and XIAP, are currently being evaluated.
Hepatocellular carcinoma (HCC) is the third most common cause of cancer related death worldwide. Vitamin D (VD) has been implicated in the prevention of multiple cancers- some with inactivation of TGF-β signaling. Recently, inactivation of TGF- β signaling has been associated with HCC. We analyzed whole genome data for TGF- βsignaling and examined the role of VD in the context of TGF- β inactivation in HCC. Methods: Databases of HCC Genomics (COSMIC) and transcriptomics (TCGA) were analyzed. The effect of calcitriol on cell proliferation was measured in HCC cell lines; Hep-G2 and MEF cells, with knocked down TGF-β/β2-spectrin (β2SP). Wild-type (WT), Sptbn1+/- and Smad3+/- mice were fed with diets containing 200 IU VD/kg or 10,000 IU VD/kg for 9 weeks. Hepatocyte proliferation was analyzed through Ki67 staining. The expression of cyclin D1 was evaluated by Western blotting. A whole genome gene expression profiling array was performed from liver samples of those mice using Illumina MouseWG-6 v2.0 gene expression arrays®. Lastly, liver samples from patients with HCC who had received VD supplementation were evaluated by immunohistochemistry. Results: Whole genome data revealed aberrant TGF- β signaling in ~70% of HCCs. Treatment with calcitriol suppressed the growth of HCC cell lines, regardless of their TGF-β pathway status. Inhibition of proliferation was noted in WT MEFs as well as in the Sptbn1+/- and Sptbn1-/- cells. High dose VD reduced hepatic proliferation and cyclin D1 levels in Sptbn1+/- and Smad3+/- mutants, and in WT mice. The microarray data demonstrated the most significant changes in gene expression were associated with acute phase inflammatory response (p<0.005). Validation studies showed a significant fold change difference of P-PARA, OAZ1, TLR7 and TLR 9 after high dose VD diet between WT mice and the mutants (p<0.05). VD supplementation to patients with HCC was associated with higher expression of β2SP (p<.0001) and TβRII (p=.013) compared with that of patients who did not receive the supplement. Conclusions: Whole genome data revealed aberrant TGF- β signaling in ~ 70% of HCCs. VD suppresses the proliferation of HCC cell lines as well as normal hepatocytes potentially through restoring of TGF- β tumor suppressor signaling. VD modulates inflammation markedly differently in the setting of TGF- βsignaling inactivation. Taken together, these results suggest that VD treatment strategies could potentially be pivotal in prevention and treatment of HCC. 770 Sulfatase2 (SULF2) Promotes Angiogenesis in Hepatocellular Carcinoma Partly Through the TGFβ1/Periostin Signaling Pathway Gang Chen, Alexander Miamen, Renumathy Dhanasekaran, Catherine D. Moser, Shaoshan Han, Yichun Chen, Yong Fang, Dongye Yang, Hassan M. Shaleh, Chunling Hu, Snorri Thorgeirsson, Lewis R. Roberts Background/Aim: The growth of hepatocellular carcinoma (HCC) requires the formation of new blood vessels, which has provided a strong rationale for antiangiogenic strategies for therapy of HCC. However, the molecular pathways involved in HCC angiogenesis are
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incompletely characterized. The heparin-degrading endosulfatase 2, SULF2, affects the activity of a number of heparin-binding growth factors by removing 6-O-sulfates from heparan sulfate proteoglycans (HSPGs). In our previous study, we showed that SULF2 can increase HCC growth and that high SULF2 expression predicts a worse prognosis after resection. HSPGs are known to mediate angiogenesis. Therefore, we investigated whether SULF2 can promote angiogenesis of HCC and explored its mechanism. Methods: The correlation between SULF2 mRNA and angiogenesis associated gene mRNAs was assessed in 139 resected HCCs by cDNA microarray analysis. Angiogenesis associated gene expression and microvascular density were assessed in diethyl nitrosamine (DEN)-induced HCC tumors in SULF2 transgenic mice as well as in xenografts established from SULF2-transfected Hep3B HCC cells. Cell viability, cell migration and tube formation ability of human hepatic sinusoidal endothelial cells (HHSEC) and human umbilical vein endothelial cells (HUVEC) were assessed after co-culture with SULF2-negative Hep3B cells transfected with SULF2 or SULF2-expressing Huh7 cells transfected with shRNA targeting SULF2. Angiogenic signaling pathways were assayed by Western blotting. Results: SULF2 mRNA expression was positively correlated with key angiogenesis associated genes which include CD31, VACAM1, VEGF B, and SDF1 in 139 resected HCCs. Microvascular density was increased in tumor tissues from both SULF2 up-regulated mouse models. Upregulation of SULF2 in Hep3B cells increased cell viability, cell migration and tube formation ability of HHSEC and HUVEC in a paracrine fashion; conversely knockdown of SULF2 in Huh7 cells reversed these morphologic features. Assessment of angiogenic pathways showed that the periostin pathway was activated in HHSEC cells after co-culture with SULF2 up-regulated Hep3B cells. The downstream factors p-AKT and p-FAK were also increased. The changes in p-AKT and p-FAK were further enhanced by activation of the TGF- β1 signaling pathway. The same association of TGF- β1, periostin, and p-FAK signaling changes was observed in both SULF2 up-regulated mouse HCC tumor models. Conclusions: Together, these findings suggest that SULF2 promotes angiogenesis of HCCs by increasing the angiogenic potency of HHSEC in a paracrine fashion. The TGFβ1/periostin signaling pathway plays an important role in regulating SULF2mediated angiogenesis of HCC. The SULF2/TGF β1/periostin pathway may be a rational target for anti-angiogenic therapy of HCC.
PLINK was used for statistical analysis. Results: Sequencing of ABCB4 identified 1 splice site, 9 Synonymous and 9 NS variants. Of note, one NS variant, p.A934T, described in LPAC patients, was present in 4 subjects in the ET group and none in the SNL group. All 4 affected individuals were of African American (AA) race (20 AA in the ET and 13 AA in the SNL group). Population analyses indicate that p.A934T is differentially represented in US racial subgroups, with an AA heterozygote frequency of 2.45%, compared with <0.01% among European Americans. Armitage trend test demonstrated p.A934T is significantly associated with ET outcome when examining all races (p=0.04). Other variants reported in LPAC or PFIC3 patients (p.T34M, p.T175A, p.R590Q, p.R652G, p.R788Q, p.E1058K) were similarly distributed among ET and SNL groups. Conclusions: This is the first WES study to discover genetic determinants of clinical outcomes in BA and identified ABCB4 as a biologically plausible candidate gene modifier. Heterozygosity for p.A934T is overrepresented among AA subjects with BA requiring early transplantation post-HPE. This finding underscores the utility of WES and subsequent validation studies of one candidate gene in a large, well-phenotyped cohort of racially diverse subjects with BA. With further application, this approach is poised to discover additional genetic contributors to BA outcomes. Support: NIH (TL1TR000456, U01DK062456, U01DK062470) & the Bauer, Spain & Alpard Foundations 846 Mutations in Tight Junction Protein 2 Underlie a Spectrum of Cholestatic Liver Disease Melissa Sambrotta, Sandra Strautnieks, Laura Brett, Suzanne Davison, Richard J. Thompson Background. Tight Junction Protein 2 is a cytoplasmic protein essential for the assembly of cell-cell junctional structures. It is encoded by TJP2. In 2003 a single, incompletely penetrant, missense mutation was identified in TJP2 in Amish patients with hypercholanaemia, in the absence of significant liver disease. We have recently shown that homozygous protein-truncating mutations in TJP2 are present in patients with severe early-onset cholestatic liver disease. Most of these patients required liver transplantation. Aim and Methods. In order to explore the wider phenotypic spectrum associated with mutations in TJP2, a cohort of 53 patients with severe early-onset of normal GGT cholestasis was selected. No mutations in the known progressive familial intrahepatic cholestasis genes ( ABCB11 and ATP8B1) had been identified. Genetic testing was undertaken using a combination of next-generation sequencing technology and Sanger DNA sequencing. Pathogenicity of missense mutations was determined with in silico analysis. Results. Genetic analysis revealed homozygous mutations in 8 patients. Three deletions and 2 nonsense mutation were identified, all predicted to lead to the truncation of the protein. In three families missense mutations were found. p.Gly737Arg was present in one patient who required liver transplantation at 13 months. However p.His788Leu was found in 2 unrelated families. Both patients had severe earlyonset cholestasis, which subsequently remitted. One of these patients relapsed at 5 years, but remitted again at age 7 years. Conclusion. We have previously described 8 families, including 12 individuals, with 8 different protein truncating mutations. We now report a further 8 families. Six manifest persistent cholestasis: five had protein-truncating mutations, and one had a homozygous missense mutation. The other 2 individuals had remitting cholestasis and were both homozygous for the same missense mutation. TJP2 was previously shown to underlie a condition with minimal liver disease. In addition to our recent identification of mutations in patients with progressive familial intrahepatic cholestasis, we now show that it can also lead to an intermediate phenotype. It is therefore clear that TJP2 deficiency represents a spectrum of liver disease phenotypes.
771 Mitochondria-Targeting Antioxidant Attenuates Cholestasis-Induced Liver Injury in Mice Yanjun Shi, Hasibur Rehman, Yasodha Krishnasamy, Venkat K. Ramshesh, Rick G. Schnellmann, John J. Lemasters, Michael P. Murphy, Zhi Zhong Background: Our previous study showed that the mitochondrial permeability transition (MPT) occurs in the liver after cholestasis, leading to cell death. Aim: Because reactive oxygen species (ROS) are a known stimulator of the MPT, we investigated whether MitoQ, an orally active antioxidant that accumulates in mitochondria, attenuates cholestatic liver injury caused by bile duct ligation (BDL) in mice. Methods: MitoQ (500 μM) was added to drinking water. Three days after starting MitoQ feeding, mice were subjected to BDL, and livers were harvested 1 and 3 days later. Mitochondrial depolarization and cell death were detected in living mice by intravital confocal/multiphon microscopy of rhodamine 123 (Rh123) and propidium iodide (PI), respectively, and onset of the MPT was assessed by entry of calcein into mitochondria. Results: 4-Hydroxynonenal adducts (4-HNE) increased markedly after BDL, indicating ROS formation, and MitoQ blunted increases in 4-HNE. Rh123 fluorescence was punctate in virtually all hepatocytes in control mice, indicating mitochondrial polarization. At 6 h after BDL, Rh123 fluorescence disappeared in many hepatocytes (~18/hpf) whereas PI-positive non-viable hepatocytes were <1/hpf, indicating mitochondrial depolarization prior to cell death. Calcein resided in the cytosol in hepatocytes of sham-operated mice but entered mitochondria 6 h after BDL, confirming the onset of the MPT in vivo. MitoQ decreased the number of depolarized hepatocytes after BDL by 72%. Serum ALT increased from 46 U/L before surgery to 3300 and 1700 U/L at 1 and 3 days, respectively, after BDL. ALT was blunted by 85% 1 day after BDL in mice given MitoQ. Focal necrosis occurred in 17% of liver tissue 1 day after BDL, and MitoQ attenuated necrosis to 5%. TUNEL-positive cells were barely detectable in livers after sham operation but increased to 4% and 2% at 1 and 3 days, respectively, after BDL and MitoQ decreased TUNEL-positive cells to 0.5%. Cleaved caspase-3 was barely detectable in livers from shamoperated mice, increased markedly after BDL, and was decreased by MitoQ. Conclusion. Taken together, we suggest that MitoQ decreases liver injury after BDL by blocking ROSinduced MPT onset. This mitochondria-targeting antioxidant may be a promising therapy to prevent early stage cholestatic liver injury (NIDDK).
847 The Types of Inflammatory Bowel Disease (IBD) Predispose to Distinct Clinical Phenotypes of Primary Sclerosing Cholangitis (PSC) in Children Keaton R. Jones, Philip Bufler, Alexander G. Miethke Background: PSC is a chronic progressive cholangiopathy which is commonly associated with IBD. Since the gut-liver axis is considered to play an important role in the pathogenesis of PSC we hypothesized that the type of IBD determined the disease course of pediatric onset PSC. Methods: In this retrospective study, medical records of 52 patients who received medical care for PSC at a single institution between 2009 and 2013 were reviewed. Results: The clinical diagnosis of PSC was confirmed in all 52 subjects based on review of reports of hepatobiliary imaging and liver biopsy. A diagnosis of IBD was established by endoscopy in 43 (83%), comprising of Crohn disease (CD) in 16 (31%) and ulcerative colitis (UC) in 27 (52%) patients. Ethnicity was primarily Caucasian in both groups (81 vs 77% in CD vs UC), but males predominated in the CD group (62 vs 46% males in CD vs UC). Mean age of diagnosis of PSC was 10.8 years in CD-PSC and 12.0 in UC-PSC patients. CD and PSC were often diagnosed simultaneously whereas the diagnosis of UC preceded detection of abnormal liver enzymes by a mean of 7 months. Maintenance therapy of colitis differed between both groups in regards to use of immunomodulator and corticosteroid therapy. Infliximab was used in 43% of CD- and 27 % of UC-PSC subjects. 9 patients (7 with PSCUC) with end-stage liver disease were referred for liver transplantation and were excluded from subsequent analysis. Of 34 patients with PSC, IBD and native liver, 6 (18%) were found to have autoimmune sclerosing cholangitis based on positive ANA serology and liver histomorphology with interface hepatitis (5 in UC- group). For 18 subjects with laboratory values available at the time of diagnosis of PSC, serum GGT levels and white blood cell counts (WBC) were significantly higher in CD- than in UC patients (see table). During follow up, peak serum total bilirubin levels and those obtained at the most recent visit were higher in CD- PSC patients. Importantly, elevated levels of biomarkers of cholestasis were associated with a higher prevalence of extrahepatic biliary disease in CD-PSC patients. Narrowing or dilatation with caliber change affecting the common hepatic or common bile ducts were detected by ERCP or MRCP in 9/12 PSC-CD (75%) compared with 7/ 21 PSCUC patients (33%; p=0.03). Conclusion: Compared with UC, concomitant CD predisposes in pediatric onset PSC to extrahepatic bile duct injury and greater degree of cholestasis, as gauged by serum biochemistries. How disease pathogenesis and treatment of colitis are linked to short and long-term outcomes in CD- and UC-PSC patients, especially progression of bile duct epithelial injury and biliary fibrosis, requires further investigation.
845 Whole Exome Sequencing Identifies ABCB4 Gene Variants As Modifiers of Biliary Atresia Outcomes Anya Mezina, Khanjan Gandhi, Aniko Sabo, Donna Muzny, Richard Gibbs, Madhuri Hegde, Saul J. Karpen Background: The role of genetic factors in determining clinical outcomes in biliary atresia (BA) after Kasai hepatoportoenterostomy (HPE) are unknown. To begin to address this, we performed a pilot study of whole exome sequencing (WES) in 20 BA patients from the NIDDK-supported ChiLDREN dataset to identify variants in genes that stratify between two disparate outcome groups: 10 who underwent early transplant prior to age 2 (ET) compared to 10 who survived beyond age 4 with native liver (SNL) and normal platelet count (>150,000). WES identified ~ 50 candidate genes with disproportionately more non-synonymous (NS) variants in the ET compared to SNL group. Among this list was the ABCB4 phospholipid floppase gene, which, if verified, would provide a pathophysiological link to worsening cholangiopathy in BA since ABCB4 gene mutations are implicated in liver diseases such as Low Phospholipid Associated Cholelithiasis (LPAC) and Progressive Familial Intrahepatic Cholestasis 3 (PFIC3). We hypothesized that deleterious variants in ABCB4 would modulate phenotypic severity and sought to validate this in a larger cohort of ET and SNL ChiLDREN subjects. Methods: Sanger DNA sequencing was performed for all 27 coding exons and the promoter region of the ABCB4 gene from 195 ChiLDREN subjects stratified as SNL (n=97) or ET (n=98). Variants were called by comparison to reference NM_018849.2.
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