LRH-1 agonist DLPC through STAT6 promotes macrophage polarization and prevents parenteral nutrition-associated cholestasis in mice.

BACKGROUND AND AIMS: Parenteral nutrition-associated cholestasis (PNAC) is an important complication in patients with intestinal failure with reduced LRH-1 expression. Here, we hypothesized that LRH-1 activation by its agonist, dilauroylphosphatidylcholine (DLPC), would trigger signal transducer and activator of transcription 6 (STAT6) signaling and hepatic macrophage polarization that would mediate hepatic protection in PNAC. APPROACH AND RESULTS: PNAC mouse model (oral DSSx4d followed by PNx14d; DSS-PN) was treated with LRH-1 agonist DLPC (30 mg/kg/day) intravenously. DLPC treatment prevented liver injury and cholestasis while inducing hepatic mRNA expression of Nr5a2 (nuclear receptor subfamily 5 group A member 2), Abcb11 (ATP binding cassette subfamily B member 11), Abcg5 (ATP-binding cassette [ABC] transporters subfamily G member 5), Abcg8 (ATP-binding cassette [ABC] transporters subfamily G member 8), nuclear receptor subfamily 0, and ATP-binding cassette subfamily C member 2 ( Abcc2) mRNA, all of which were reduced in PNAC mice. To determine the mechanism of the DLPC effect, we performed RNA-sequencing analysis of the liver from Chow, DSS-PN, and DSS-PN/DLPC mice, which revealed DLPC upregulation of the anti-inflammatory STAT6 pathway. In intrahepatic mononuclear cells or bone-marrow derived macrophages (BMDM) from PNAC mice, DLPC treatment prevented upregulation of pro-inflammatory (M1) genes, suppressed activation of NFκB and induced phosphorylation of STAT6 and its target genes, indicating M2 macrophage polarization. In vitro, incubation of DLPC with cultured macrophages showed that the increased Il-1b and Tnf induced by exposure to lipopolysaccharides or phytosterols was reduced significantly, which was associated with increased STAT6 binding to promoters of its target genes. Suppression of STAT6 expression by siRNA in THP-1 cells exposed to lipopolysaccharides, phytosterols, or both resulted in enhanced elevation of IL-1B mRNA expression. Furthermore, the protective effect of DLPC in THP-1 cells was abrogated by STAT6 siRNA. CONCLUSIONS: These results indicate that activation of LRH-1 by DLPC may protect from PNAC liver injury through STAT6-mediated macrophage polarization.

Diagnostic accuracy of serum matrix metalloproteinase-7 as a biomarker of biliary atresia in a large North American cohort.

BACKGROUND AND AIMS: High levels of serum matrix metalloproteinase-7 (MMP-7) have been linked to biliary atresia (BA), with wide variation in concentration cutoffs. We investigated the accuracy of serum MMP-7 as a diagnostic biomarker in a large North American cohort. APPROACH AND RESULTS: MMP-7 was measured in serum samples of 399 infants with cholestasis in the Prospective Database of Infants with Cholestasis study of the Childhood Liver Disease Research Network, 201 infants with BA and 198 with non-BA cholestasis (age median: 64 and 59 days, p = 0.94). MMP-7 was assayed on antibody-bead fluorescence (single-plex) and time resolved fluorescence energy transfer assays. The discriminative performance of MMP-7 was compared with other clinical markers. On the single-plex assay, MMP-7 generated an AUROC of 0.90 (CI: 0.87-0.94). At cutoff 52.8 ng/mL, it produced sensitivity = 94.03%, specificity = 77.78%, positive predictive value = 64.46%, and negative predictive value = 96.82% for BA. AUROC for gamma-glutamyl transferase = 0.81 (CI: 0.77-0.86), stool color = 0.68 (CI: 0.63-0.73), and pathology = 0.84 (CI: 0.76-0.91). Logistic regression models of MMP-7 with other clinical variables individually or combined showed an increase for MMP-7+gamma-glutamyl transferase AUROC to 0.91 (CI: 0.88-0.95). Serum concentrations produced by time resolved fluorescence energy transfer differed from single-plex, with an optimal cutoff of 18.2 ng/mL. Results were consistent within each assay technology and generated similar AUROCs. CONCLUSIONS: Serum MMP-7 has high discriminative properties to differentiate BA from other forms of neonatal cholestasis. MMP-7 cutoff values vary according to assay technology. Using MMP-7 in the evaluation of infants with cholestasis may simplify diagnostic algorithms and shorten the time to hepatoportoenterostomy.

Establishing Neonate-specific Prognostic Markers in Acute Liver Failure: Admission Alpha Fetoprotein and Novel Neonatal Acute Liver Failure Scores Predict Patient Outcomes.

OBJECTIVE: To develop neonate-specific prediction models for survival with native liver (SNL) in neonatal acute liver failure (ALF) and to determine if these prediction models have superior accuracy to existing models for older children with ALF. STUDY DESIGN: A single-center, retrospective chart review was conducted on neonates ≤ 30 days of life between 2005 and 2022 with ALF (international normalized ratio ≥ 2 or prothrombin time ≥ 20s and liver dysfunction). Statistical analysis included comparison of patients by outcome of SNL and generalized linear modeling to derive prediction models. The predictive accuracy of variables was evaluated by receiver operating characteristic (ROC) analysis and Kaplan-Meier survival analysis. RESULTS: A total of 51 patients met inclusion criteria. The most common causes of neonatal ALF included ischemia (22%), infection (20%), and gestational alloimmune liver disease (16%). Overall SNL rate was 43% (n = 22). Alpha fetoprotein levels were higher in SNL patients (P = .034) and differed more significantly by SNL status among nongestational alloimmune liver disease patients (n = 21, P = .001). An alpha fetoprotein < 4775 ng/mL had 75% sensitivity and 100% specificity to predict death or transplant in nongestational alloimmune liver disease patients with an area under the ROC curve of 0.81. A neonate-specific admission model (international normalized ratio and ammonia) and peak model (prothrombin time and ammonia) also predicted SNL with good accuracy (area under the ROC curve = 0.73 and 0.82, respectively). CONCLUSIONS: We identified neonate-specific prognostic variables for SNL in ALF. Findings from our study may help early risk stratification to guide medical decision-making and consideration for liver transplantation.

Predictors of 6-year event-free survival in Alagille syndrome patients treated with maralixibat, an ileal bile acid transporter inhibitor.

BACKGROUND AND AIMS: Refractory pruritus and other complications of cholestasis are indications for liver transplantation (LT) in patients with Alagille syndrome (ALGS). We evaluated predictors of event-free survival and transplant-free survival in patients with ALGS treated with maralixibat (MRX), an ileal bile acid transporter inhibitor. APPROACH AND RESULTS: We assessed patients with ALGS from 3 clinical trials of MRX with up to 6 years of follow-up. Event-free survival was defined as the absence of LT, surgical biliary diversion, hepatic decompensation, or death; transplant-free survival was the absence of LT or death. Forty-three potential predictors were evaluated, including age, pruritus (ItchRO[Obs] 0-4 scale), biochemistries, platelets, and serum bile acids. Harrell's concordance statistic assessed goodness-of-fit, and then, Cox proportional hazard models confirmed the statistical significance of the predictors identified. A further analysis was performed to identify cutoffs using a grid search. Seventy-six individuals met the criteria of receiving MRX for ≥48 weeks with laboratory values available at week 48 (W48). The median duration of MRX was 4.7 years (IQR: 1.6-5.8); 16 had events (10 LT, 3 decompensation, 2 death, and 1 surgical biliary diversion). The 6-year event-free survival improved with a clinically meaningful >1-point ItchRO(Obs) reduction from baseline to W48 (88% vs. 57%; p = 0.005), W48 bilirubin < 6.5 mg/dL (90% vs. 43%; p < 0.0001), and W48 serum bile acid < 200 µmol/L (85% vs. 49%; p = 0.001). These parameters were also predictive of 6-year transplant-free survival. CONCLUSIONS: Improvement in pruritus by 48 weeks, and lower W48 bilirubin and serum bile acid levels were associated with fewer events. These data may help identify potential markers of disease progression for ALGS patients treated with MRX.

Serum bile acids as a prognostic biomarker in biliary atresia following Kasai portoenterostomy.

BACKGROUND AND AIMS: In biliary atresia, serum bilirubin is commonly used to predict outcomes after Kasai portoenterostomy (KP). Infants with persistently high levels invariably need liver transplant, but those achieving normalized levels have a less certain disease course. We hypothesized that serum bile acid levels could help predict outcomes in the latter group. APPROACH AND RESULTS: Participants with biliary atresia from the Childhood Liver Disease Research Network were included if they had normalized bilirubin levels 6 months after KP and stored serum samples from the 6-month post-KP clinic visit ( n  = 137). Bile acids were measured from the stored serum samples and used to divide participants into ≤40 µmol/L ( n  = 43) or >40 µmol/L ( n  = 94) groups. At 2 years of age, the ≤40 µmol/L compared with >40 µmol/L group had significantly lower total bilirubin, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, bile acids, and spleen size, as well as significantly higher albumin and platelet counts. Furthermore, during 734 person-years of follow-up, those in the ≤40 µmol/L group were significantly less likely to develop splenomegaly, ascites, gastrointestinal bleeding, or clinically evident portal hypertension. The ≤40 µmol/L group had a 10-year cumulative incidence of liver transplant/death of 8.5% (95% CI: 1.1%-26.1%), compared with 42.9% (95% CI: 28.6%-56.4%) for the >40 µmol/L group ( p  = 0.001). CONCLUSIONS: Serum bile acid levels may be a useful prognostic biomarker for infants achieving normalized bilirubin levels after KP.

Serum biomarkers correlated with liver stiffness assessed in a multicenter study of pediatric cholestatic liver disease.

BACKGROUND AND AIMS: Detailed investigation of the biological pathways leading to hepatic fibrosis and identification of liver fibrosis biomarkers may facilitate early interventions for pediatric cholestasis. APPROACH AND RESULTS: A targeted enzyme-linked immunosorbent assay-based panel of nine biomarkers (lysyl oxidase, tissue inhibitor matrix metalloproteinase (MMP) 1, connective tissue growth factor [CTGF], IL-8, endoglin, periostin, Mac-2-binding protein, MMP-3, and MMP-7) was examined in children with biliary atresia (BA; n = 187), alpha-1 antitrypsin deficiency (A1AT; n = 78), and Alagille syndrome (ALGS; n = 65) and correlated with liver stiffness (LSM) and biochemical measures of liver disease. Median age and LSM were 9 years and 9.5 kPa. After adjusting for covariates, there were positive correlations among LSM and endoglin ( p = 0.04) and IL-8 ( p < 0.001) and MMP-7 ( p < 0.001) in participants with BA. The best prediction model for LSM in BA using clinical and lab measurements had an R2 = 0.437; adding IL-8 and MMP-7 improved R2 to 0.523 and 0.526 (both p < 0.0001). In participants with A1AT, CTGF and LSM were negatively correlated ( p = 0.004); adding CTGF to an LSM prediction model improved R2 from 0.524 to 0.577 ( p = 0.0033). Biomarkers did not correlate with LSM in ALGS. A significant number of biomarker/lab correlations were found in participants with BA but not those with A1AT or ALGS. CONCLUSIONS: Endoglin, IL-8, and MMP-7 significantly correlate with increased LSM in children with BA, whereas CTGF inversely correlates with LSM in participants with A1AT; these biomarkers appear to enhance prediction of LSM beyond clinical tests. Future disease-specific investigations of change in these biomarkers over time and as predictors of clinical outcomes will be important.

Pharmacologic activation of hepatic farnesoid X receptor prevents parenteral nutrition-associated cholestasis in mice.

BACKGROUND AND AIMS: Parenteral nutrition (PN)-associated cholestasis (PNAC) complicates the care of patients with intestinal failure. In PNAC, phytosterol containing PN synergizes with intestinal injury and IL-1ß derived from activated hepatic macrophages to suppress hepatocyte farnesoid X receptor (FXR) signaling and promote PNAC. We hypothesized that pharmacological activation of FXR would prevent PNAC in a mouse model. APPROACH AND RESULTS: To induce PNAC, male C57BL/6 mice were subjected to intestinal injury (2% dextran sulfate sodium [DSS] for 4 days) followed by central venous catheterization and 14-day infusion of PN with or without the FXR agonist GW4064. Following sacrifice, hepatocellular injury, inflammation, and biliary and sterol transporter expression were determined. GW4064 (30 mg/kg/day) added to PN on days 4-14 prevented hepatic injury and cholestasis; reversed the suppressed mRNA expression of nuclear receptor subfamily 1, group H, member 4 (Nr1h4)/FXR, ATP-binding cassette subfamily B member 11 (Abcb11)/bile salt export pump, ATP-binding cassette subfamily C member 2 (Abcc2), ATP binding cassette subfamily B member 4(Abcb4), and ATP-binding cassette subfamily G members 5/8(Abcg5/8); and normalized serum bile acids. Chromatin immunoprecipitation of liver showed that GW4064 increased FXR binding to the Abcb11 promoter. Furthermore, GW4064 prevented DSS-PN-induced hepatic macrophage accumulation, hepatic expression of genes associated with macrophage recruitment and activation (ll-1b, C-C motif chemokine receptor 2, integrin subunit alpha M, lymphocyte antigen 6 complex locus C), and hepatic macrophage cytokine transcription in response to lipopolysaccharide in vitro. In primary mouse hepatocytes, GW4064 activated transcription of FXR canonical targets, irrespective of IL-1ß exposure. Intestinal inflammation and ileal mRNAs (Nr1h4, Fgf15, and organic solute transporter alpha) were not different among groups, supporting a liver-specific effect of GW4064 in this model. CONCLUSIONS: GW4064 prevents PNAC in mice through restoration of hepatic FXR signaling, resulting in increased expression of canalicular bile and of sterol and phospholipid transporters and suppression of macrophage recruitment and activation. These data support augmenting FXR activity as a therapeutic strategy to alleviate or prevent PNAC.

Risk of variceal hemorrhage and pretransplant mortality in children with biliary atresia.

BACKGROUND AND AIMS: The natural history of gastroesophageal variceal hemorrhage (VH) in biliary atresia (BA) is not well characterized. We analyzed risk factors, incidence, and outcomes of VH in a longitudinal multicenter study. APPROACH AND RESULTS: Participants enrolled in either an incident (Prospective Database of Infants with Cholestasis [PROBE]) or prevalent (Biliary Atresia Study of Infants and Children [BASIC]) cohort of BA were included. Variceal hemorrhage (VH) was defined based on gastrointestinal bleeding in the presence of varices accompanied by endoscopic or nontransplant surgical intervention. Cumulative incidence of VH and transplant-free survival was compared based on features of portal hypertension (e.g., splenomegaly, thrombocytopenia) and clinical parameters at baseline in each cohort (PROBE: 1.5 to 4.5 months after hepatoportoenterostomy [HPE]; BASIC: at enrollment > 3 years of age). Analyses were conducted on 869 children with BA enrolled between June 2004 and December 2020 (521 in PROBE [262 (51%) with a functioning HPE] and 348 in BASIC). The overall incidence of first observed VH at 5 years was 9.4% (95% CI: 7.0-12.4) in PROBE and 8.0% (5.2-11.5) in BASIC. Features of portal hypertension, platelet count, total bilirubin, aspartate aminotransferase (AST), albumin, and AST-to-platelet ratio index at baseline were associated with an increased risk of subsequent VH in both cohorts. Transplant-free survival at 5 years was 45.1% (40.5-49.6) in PROBE and 79.2% (74.1-83.4) in BASIC. Two (2.5%) of 80 participants who had VH died, whereas 10 (12.5%) underwent transplant within 6 weeks of VH. CONCLUSIONS: The low risk of VH and associated mortality in children with BA needs to be considered in decisions related to screening for varices and primary prophylaxis of VH.

miR-199a-5p inhibits the expression of ABCB11 in obstructive cholestasis.

ATP-binding cassette, subfamily B member 11 (ABCB11) is an efflux transporter for bile acids on the liver canalicular membrane. The expression of this transporter is reduced in cholestasis; however, the mechanisms contributing to this reduction are unclear. In this study, we sought to determine whether miR-199a-5p contributes to the depletion of ABCB11/Abcb11 in cholestasis in mice. In a microRNA (miRNA) screen of mouse liver after common bile duct ligation (CBDL), we found that miR-199a-5p was significantly upregulated by approximately fourfold. In silico analysis predicted that miR-199a-5p would target the 3'-untranslated region (3'-UTR) of ABCB11/Abcb11 mRNA. The expression of ABCB11-3'-UTR luciferase construct in Huh-7 cells was markedly inhibited by cotransfection of a miRNA-199a-5p mimic, which was reversed by an miRNA-199a-5p mimic inhibitor. We also show treatment of mice after CBDL with the potent nuclear receptor FXR agonist obeticholic acid (OCA) significantly increased Abcb11 mRNA and protein and decreased miR-199a-5p expression. Computational mapping revealed a well-conserved FXR-binding site (FXRE) in the promoter of the gene encoding miR-199a-5, termed miR199a-2. Electromobility shift, chromatin immunoprecipitation, and miR199a-2 promoter-luciferase assays confirmed that this binding site was functional. Finally, CBDL in mice led to depletion of nuclear repressor NcoR1 binding at the miR199a-2 promoter, which facilitates transcription of miR199a-2. In CBDL mice treated with OCA, NcoR1 recruitment to the miR199a-2 FXRE was maintained at levels found in sham-operated mice. In conclusion, we demonstrate that miR-199a-5p is involved in regulating ABCB11/Abcb11 expression, is aberrantly upregulated in obstructive cholestasis, and is downregulated by the FXR agonist OCA.

Malnutrition in Biliary Atresia: Assessment, Management, and Outcomes.

Children with biliary atresia (BA), particularly infants, are at high risk for malnutrition attributed to a multitude of factors, including poor oral intake and intolerance of enteral feeding, fat malabsorption, abnormal nutrient metabolism, and increased caloric demand. Malnutrition and sarcopenia negatively impact outcomes in BA, leading to higher pretransplant and posttransplant morbidity and mortality. This review summarizes factors contributing to nutritional deficiencies in BA and offers an organized approach to the assessment and management of malnutrition in this vulnerable population.

NF-κB Regulation of LRH-1 and ABCG5/8 Potentiates Phytosterol Role in the Pathogenesis of Parenteral Nutrition-Associated Cholestasis.

BACKGROUND AND AIMS: Chronically administered parenteral nutrition (PN) in patients with intestinal failure carries the risk for developing PN-associated cholestasis (PNAC). We have demonstrated that farnesoid X receptor (FXR) and liver X receptor (LXR), proinflammatory interleukin-1 beta (IL-1ß), and infused phytosterols are important in murine PNAC pathogenesis. In this study we examined the role of nuclear receptor liver receptor homolog 1 (LRH-1) and phytosterols in PNAC. APPROACH AND RESULTS: In a C57BL/6 PNAC mouse model (dextran sulfate sodium [DSS] pretreatment followed by 14 days of PN; DSS-PN), hepatic nuclear receptor subfamily 5, group A, member 2/LRH-1 mRNA, LRH-1 protein expression, and binding of LRH-1 at the Abcg5/8 and Cyp7a1 promoter was reduced. Interleukin-1 receptor-deficient mice (Il-1r-/- /DSS-PN) were protected from PNAC and had significantly increased hepatic mRNA and protein expression of LRH-1. NF-κB activation and binding to the LRH-1 promoter were increased in DSS-PN PNAC mice and normalized in Il-1r-/- /DSS-PN mice. Knockdown of NF-κB in IL-1ß-exposed HepG2 cells increased expression of LRH-1 and ABCG5. Treatment of HepG2 cells and primary mouse hepatocytes with an LRH-1 inverse agonist, ML179, significantly reduced mRNA expression of FXR targets ATP binding cassette subfamily C member 2/multidrug resistance associated protein 2 (ABCC2/MRP2), nuclear receptor subfamily 0, groupB, member 2/small heterodimer partner (NR0B2/SHP), and ATP binding cassette subfamily B member 11/bile salt export pump (ABCB11/BSEP). Co-incubation with phytosterols further reduced expression of these genes. Similar results were obtained by suppressing the LRH-1 targets ABCG5/8 by treatment with small interfering RNA, IL-1ß, or LXR antagonist GSK2033. Liquid chromatography-mass spectrometry and chromatin immunoprecipitation experiments in HepG2 cells showed that ATP binding cassette subfamily G member 5/8 (ABCG5/8) suppression by GSK2033 increased the accumulation of phytosterols and reduced binding of FXR to the SHP promoter. Finally, treatment with LRH-1 agonist, dilauroyl phosphatidylcholine (DLPC) protected DSS-PN mice from PNAC. CONCLUSIONS: This study suggests that NF-κB regulation of LRH-1 and downstream genes may affect phytosterol-mediated antagonism of FXR signaling in the pathogenesis of PNAC. LRH-1 could be a potential therapeutic target for PNAC.

Macronutrient and Micronutrient Intake in Adolescents With Non-alcoholic Fatty Liver Disease: The Association With Disease Severity.

OBJECTIVES: While dietary changes are recommended to treat pediatric non-alcoholic fatty liver disease (NAFLD), the role of specific nutrients in disease progression is unclear. The objective of this study is to (1) assess the macronutrient and micronutrient intake in adolescents with liver biopsy proven NAFLD [with and without non-alcoholic steatohepatitis (NASH)] and lean controls; (2) determine nutritional predictors of disease severity amongst these groups. METHODS: Adolescents with biopsy-proven NAFLD and lean controls completed the Harvard Food Frequency Questionnaire. RESULTS: Twenty-eight NAFLD and 15 lean controls were studied. NAFLD with (n = 20) and without NASH (n = 8) had similar total calorie, protein, fat, and carbohydrate intake. Subjects with NASH had higher total sugar (122.3 ± 48.3 vs 83.1 ± 38.8 g), glucose (24.3 ± 9.3 vs 15.2 ± 7.5 g), sucrose (42.3 ± 16.9 vs 28.8 ± 11.7 g), and fructose (29.4 ± 12.5 vs 18.1 ± 8.0 g) intake than those with NAFLD but without NASH ( P < 0.05). Both NAFLD groups had similar micronutrient intake. Alanine aminotransferase (ALT) correlated with total caloric intake ( ρ = 0.4; P = 0.04). Total carbohydrate calories correlated with a higher NAS summary score ( ρ = 0.38; P = 0.04) and lobular inflammation ( ρ = 0.50; P = 0.007). Percent calories from added sugar and glucose correlated with worsening NAS summary score ( ρ = 0.44, P = 0.02; ρ = 0.48, P = 0.009) and lobular inflammation ( ρ = 0.51, P = 0.006; ρ = 0.53, P = 0.004). Percent calories from fructose correlated with lobular inflammation ( ρ = 0.56; P = 0.002). Total daily calories, protein, fat, carbohydrate, and micronutrient intake were similar between NAFLD and lean controls. CONCLUSIONS: NASH patients consume similar total calories, protein, and fat as those without NASH, but have significantly higher sugar intake. NAFLD and lean children, however, have similar macro/micronutrient intake. Histologic disease severity correlates with total carbohydrate and added sugar intake, supporting a role for simple sugar intake in NAFLD progression.

Neurodevelopmental Outcomes in Children With Inherited Liver Disease and Native Liver.

OBJECTIVE: To evaluate neurodevelopmental status among children with inherited cholestatic liver diseases with native liver and variables predictive of impairment. METHODS: Participants with Alagille syndrome (ALGS), progressive familial intrahepatic cholestasis (PFIC), and alpha 1 antitrypsin deficiency (A1AT) enrolled in a longitudinal, multicenter study and completed the Wechsler Preschool and Primary Scale of Intelligence-III or Intelligence Scale for Children-IV. Full Scale Intelligence Quotient (FSIQ) was analyzed continuously and categorically (>100, 85-99, 70-84, <70). Univariate linear regression was performed to study association between FSIQ and risk factors, stratified by disease. RESULTS: Two hundred and fifteen completed testing (ALGS n = 70, PFIC n = 43, A1AT n = 102); median age was 7.6 years (3.0-16.9). Mean FSIQ in ALGS was lower than A1AT (94 vs 101, P = 0.01). Frequency of FSIQ < 85 (>1 standard deviation [SD] below average) was highest in ALGS (29%) versus 18.6% in PFIC and 12.8% in A1AT, and was greater than expected in ALGS based on normal distribution (29% vs 15.9%, P = 0.003). ALGS scored significantly lower than test norms in almost all Wechsler composites; A1AT scored lower on Working Memory and Processing Speed; PFIC was not different from test norms. Total bilirubin, alkaline phosphatase, albumin, hemoglobin, and parental education were significantly associated with FSIQ. CONCLUSIONS: Patients with ALGS are at increased risk of lower FSIQ, whereas our data suggest A1AT and PFIC are not. A1AT and ALGS appear vulnerable to working memory and processing speed deficits suggestive of attention/executive function impairment. Malnutrition, liver disease severity, and sociodemographic factors appear related to FSIQ deficits, potentially identifying targets for early interventions.

New Insights Into Intestinal Failure-Associated Liver Disease in Children.

Development of intestinal failure-associated liver disease (IFALD) is a common complication of long-term parenteral nutrition (PN) in children and adults. The molecular and cellular mechanisms and the phases of IFALD are now being delineated. Components of PN lipid emulsions, including plant sterols, interact with hepatic innate immune activation promoted by products of gut bacterial overgrowth/dysbiosis and altered intestinal barrier function (gut-liver axis) and by episodes of sepsis to cause cholestasis and IFALD. New therapeutic strategies, including modifications of intravenous lipid emulsions to reduce pro-inflammatory fatty acids and plant sterol content, can lower the risk of IFALD, reverse cholestasis, and reduce complications, although the significance of persisting hepatic fibrosis is unknown. This review will provide an update on advances in the pathogenesis of IFALD, newer therapeutic and preventative strategies, and challenges that confront managing patients with IFALD.

Use of a Comprehensive 66-Gene Cholestasis Sequencing Panel in 2171 Cholestatic Infants, Children, and Young Adults.

OBJECTIVES: Cholestasis is caused by a wide variety of etiologies, often genetic in origin. Broad overlap in clinical presentations, particularly in newborns, renders prioritizing diagnostic investigations challenging. In this setting, a timely, comprehensive assessment using a multigene panel by a clinical diagnostic laboratory would likely prove useful. We summarize initial findings from a testing program designed to discover genetic causes of cholestasis. METHODS: A neonatal/adult sequencing panel containing 66 genes (originally 57; nine added March 2017) relevant to cholestasis was used. A broad range of eligible patients were enrolled with current/history of cholestasis without an identified cause, or unexplained chronic liver disease. DNA sequencing utilized a custom-designed capture library, and variants were classified and reported as benign, likely benign, variant of unknown significance (VOUS), likely pathogenic (LP), or pathogenic (P), according to the clinical interpretation workflow at EGL Genetics (Tucker, GA). RESULTS: A total of 2433 samples were submitted between February 2016 and December 2017; 2171 results were reported. Median turnaround time was 21 days. Results from the 2171 subjects (57% <1 year old) included 583 P variants, 79 LP variants, and 3117 VOUS; 166 P/LP variants and 415 VOUS were novel. The panel's overall diagnostic yield was 12% (n = 265/2171) representing 32 genes. The top five genetic diagnoses for the group, in order: JAG1 + NOTCH2 (Alagille syndrome), ABCB11, SERPINA1, ABCB4, and POLG. CONCLUSIONS: These findings support the utility of comprehensive rapid multigene testing in diagnosing cholestasis and highlight the evolving understanding of genetic variants contributing to the pathogenesis of cholestasis.

Presentation and Outcomes of Infants With Idiopathic Cholestasis: A Multicenter Prospective Study.

OBJECTIVES: The aim of the study was to determine the frequency and natural history of infantile idiopathic cholestasis (IC) in a large, prospective, multicenter cohort of infants. METHODS: We studied 94 cholestatic infants enrolled up to 6 months of age in the NIDDK ChiLDReN (Childhood Liver Disease Research Network) "PROBE" protocol with a final diagnosis of IC; they were followed up to 30 months of age. RESULTS: Male sex (66/94; 70%), preterm birth (22/90 with data; 24% born at < 37 weeks' gestational age), and low birth weight (25/89; 28% born at <2500 g) were frequent, with no significant differences between outcomes. Clinical outcomes included death (n = 1), liver transplant (n = 1), biochemical resolution (total bilirubin [TB] ≤1 mg/dL and ALT < 35 U/L; n = 51), partial resolution (TB > 1 mg/dL and/or ALT > 35 U/L; n = 7), and exited healthy (resolved disease per study site report but without documented biochemical resolution; n = 34). Biochemical resolution occurred at median of 9 months of age. GGT was <100 U/L at baseline in 34 of 83 participants (41%). CONCLUSIONS: Frequency of IC and of death or liver transplant was less common in this cohort than in previously published cohorts, likely because of recent discovery and diagnosis of genetic etiologies of severe/persistent cholestasis that previously were labeled as idiopathic. Preterm birth and other factors associated with increased vulnerability in neonates are relatively frequent and may contribute to IC. Overall outcome in IC is excellent. Low/normal GGT was common, possibly indicating a role for variants in genes associated with low-GGT cholestasis-this warrants further study.

Mutation Analysis and Disease Features at Presentation in a Multi-Center Cohort of Children With Monogenic Cholestasis.

OBJECTIVES: To advance our understanding of monogenic forms of intrahepatic cholestasis. METHODS: Analyses included participants with pathogenic biallelic mutations in adenosine triphosphate (ATP)-binding cassette subfamily B member 11 (ABCB11) (bile salt export pump; BSEP) or adenosine triphosphatase (ATPase) phospholipid transporting 8B1 (ATP8B1) (familial intrahepatic cholestasis; FIC1), or those with monoallelic or biallelic mutations in adenosine triphosphate (ATP)-binding cassette subfamily B member 4 (ABCB4) (multidrug resistance; MDR3), prospectively enrolled in the Longitudinal Study of Genetic Causes of Intrahepatic Cholestasis (LOGIC; NCT00571272) between November 2007 and December 2013. Summary statistics were calculated to describe baseline demographics, history, anthropometrics, laboratory values, and mutation data. RESULTS: Ninety-eight participants with FIC1 (n = 26), BSEP (n = 53, including 8 with biallelic truncating mutations [severe] and 10 with p.E297G or p.D482G [mild]), or MDR3 (n = 19, including four monoallelic) deficiency were analyzed. Thirty-five had a surgical interruption of the enterohepatic circulation (sEHC), including 10 who underwent liver transplant (LT) after sEHC. Onset of symptoms occurred by age 2 years in most with FIC1 and BSEP deficiency, but was later and more variable for MDR3. Pruritus was nearly universal in FIC1 and BSEP deficiency. In participants with native liver, failure to thrive was common in FIC1 deficiency, high ALT was common in BSEP deficiency, and thrombocytopenia was common in MDR3 deficiency. sEHC was successful after more than 1 year in 7 of 19 participants with FIC1 and BSEP deficiency. History of LT was most common in BSEP deficiency. Of 102 mutations identified, 43 were not previously reported. CONCLUSIONS: In this cohort, BSEP deficiency appears to be correlated with a more severe disease course. Genotype-phenotype correlations in these diseases are not straightforward and will require the study of larger cohorts.

Longitudinal Outcomes in Young Patients with Alpha-1-Antitrypsin Deficiency with Native Liver Reveal that Neonatal Cholestasis is a Poor Predictor of Future Portal Hypertension.

OBJECTIVES: To identify predictors of portal hypertension, liver transplantation, and death in North American youth with alpha-1-antitrypsin (AAT) deficiency, and compare with patients with AAT deficiency elsewhere. STUDY DESIGN: The Childhood Liver Disease Research Network Longitudinal Observational Study of Genetic Causes of Intrahepatic Cholestasis is a prospective, cohort study of pediatric cholestatic liver diseases, including AAT deficiency, enrolling PIZZ and PISZ subjects 0-25 years of age seen since November 2007 at 17 tertiary care centers in the US and Canada. Data from standard-of-care baseline and annual follow-up visits were recorded from medical records, history, physical examination, and laboratory studies. Participants with portal hypertension were identified based on data collected. RESULTS: We enrolled 350 participants (60% male) with a native liver; 278 (79%) entered the cohort without portal hypertension and 18 developed portal hypertension during follow-up. Thirty participants required liver transplantation; 2 patients died during 1077 person-years of follow-up. There was no difference in participants with or without preceding neonatal cholestasis progressing to transplantation or death during the study (12% vs 7%; P = .09), or in experiencing portal hypertension (28% vs 21%; P = .16); the hazard ratio for neonatal cholestasis leading to portal hypertension was P = .04. Development of portal hypertension was associated with a reduced height Z-score. CONCLUSIONS: Portal hypertension in youth with AAT deficiency impacts growth measures. Progression to liver transplantation is slow and death is rare, but the risk of complications and severe liver disease progression persists throughout childhood. A history of neonatal cholestasis is a weak predictor of severe disease.

Bone Density in Children With Chronic Liver Disease Correlates With Growth and Cholestasis.

Osteopenia and bone fractures are significant causes of morbidity in children with cholestatic liver disease. Dual-energy X-ray absorptiometry (DXA) analysis was performed in children with intrahepatic cholestatic diseases who were enrolled in the Longitudinal Study of Genetic Causes of Intrahepatic Cholestasis in the Childhood Liver Disease Research Network. DXA was performed on participants aged >5 years (with native liver) diagnosed with bile acid synthetic disorder (BASD), alpha-1 antitrypsin deficiency (A1AT), chronic intrahepatic cholestasis (CIC), and Alagille syndrome (ALGS). Weight, height, and body mass index Z scores were lowest in CIC and ALGS. Total bilirubin (TB) and serum bile acids (SBA) were highest in ALGS. Bone mineral density (BMD) and bone mineral content (BMC) Z scores were significantly lower in CIC and ALGS than in BASD and A1AT (P < 0.001). After anthropometric adjustment, bone deficits persisted in CIC but were no longer noted in ALGS. In ALGS, height-adjusted and weight-adjusted subtotal BMD and BMC Z scores were negatively correlated with TB (P < 0.001) and SBA (P = 0.02). Mean height-adjusted and weight-adjusted subtotal BMC Z scores were lower in ALGS participants with a history of bone fractures. DXA measures did not correlate significantly with biliary diversion status. Conclusion: CIC patients had significant bone deficits that persisted after adjustment for height and weight and generally did not correlate with degree of cholestasis. In ALGS, low BMD and BMC reference Z scores were explained by poor growth. Anthropometrically adjusted DXA measures in ALGS correlate with markers of cholestasis and bone fracture history. Reduced bone density in this population is multifactorial and related to growth, degree of cholestasis, fracture vulnerability, and contribution of underlying genetic etiology.