Outcomes of Childhood Cholestasis in Alagille Syndrome: Results of a Multicenter Observational Study

Alagille syndrome (ALGS) is an autosomal dominant multisystem disorder with cholestasis as a defining clinical feature. We sought to characterize hepatic outcomes in a molecularly defined cohort of children with ALGS‐related cholestasis. Two hundred and ninety‐three participants with ALGS with native liver were enrolled. Participants entered the study at different ages and data were collected retrospectively prior to enrollment, and prospectively during the study course. Genetic analysis in 206 revealed JAGGED1 mutations in 91% and NOTCH2 mutations in 4%. Growth was impaired with mean height and weight z‐scores of <−1.0 at all ages. Regression analysis revealed that every 10 mg/dL increase in total bilirubin was associated with a decrease in height z‐score by 0.10 (P = 0.03) and weight z‐score by 0.15 (P = 0.007). Total bilirubin was higher for younger participants (P = 0.03) with a median of 6.9 mg/dL for those less than 1 year old compared with a median of 1.3 mg/dL for participants 13 years or older. The median gamma glutamyl transferase also dropped from 612 to 268 in the same age groups. After adjusting for age, there was substantial within‐individual variation of alanine aminotransferase. By 20 years of age, 40% of participants had developed definite portal hypertension. Estimated liver transplant–free survival at the age of 18.5 years was 24%. Conclusions: This is the largest multicenter natural history study of cholestasis in ALGS, demonstrating a previously underappreciated burden of liver disease with early profound cholestasis, a second wave of portal hypertension later in childhood, and less than 25% of patients reaching young adulthood with their native liver. These findings will promote optimization of ALGS management and development of clinically relevant endpoints for future therapeutic trials

NorUDCA promotes degradation of α1-antitrypsin mutant Z protein by inducing autophagy through AMPK/ULK1 pathway.

Alpha-1 Antitrypsin (α1AT) Deficiency is a genetic disease in which accumulation of α1AT mutant Z (α1ATZ) protein in the ER of hepatocytes causes chronic liver injury, liver fibrosis, and hepatocellular carcinoma. No effective medical therapy is currently available for the disease. We previously found that norUDCA improves the α1AT deficiency associated liver disease by promoting autophagic degradation of α1ATZ protein in liver in a mouse model of the disease. The current study unravels the novel underlying cellular mechanism by which norUDCA modulates autophagy. HTOZ cells, modified from HeLa Tet-Off cells by transfection with the resulting pTRE1-ATZ plasmid and expressing mutant Z proteins, were studied in these experiments. The role of norUDCA in inducing autophagy, autophagy-mediated degradation of α1ATZ and the role of AMPK in norUDCA-induced autophagy were examined in the current report. NorUDCA promoted disposal of α1ATZ via autophagy-mediated degradation of α1ATZ in HTOZ cells. Activation of AMPK was required for norUDCA-induced autophagy and α1ATZ degradation. Moreover, mTOR/ULK1 was involved in norUDCA-induced AMPK activation and autophagy in HTOZ cells. Our results provide novel mechanistic insights into the therapeutic action of norUDCA in promoting the clearance of α1ATZ in vitro and suggest a novel therapeutic approach for the treatment of α1ATZ deficiency disease and its associated liver diseases

Biomarkers Associated With Future Severe Liver Disease in Children With Alpha-1-Antitrypsin Deficiency

Background and Aims: Children with alpha-1-antitrypsin deficiency (AATD) exhibit a wide range of liver disease outcomes from portal hypertension and transplant to asymptomatic without fibrosis. Individual outcomes cannot be predicted. Liver injury in AATD is caused by the accumulation in hepatocytes of the mutant Z alpha-1-antitrypsin (AAT) protein, especially the toxic, intracellular polymerized conformation. AATD patients have trace Z polymer detectable in serum with unknown significance. Methods: The Childhood Liver Disease Research Network is an NIH consortium for the study of pediatric liver diseases, including AATD. We obtained data and samples with the aim of identifying biomarkers predictive of severe AATD liver disease. Results: We analyzed prospective AATD Childhood Liver Disease Research Network data and serum samples in 251 subjects from 2007 to 2015 for outcomes and Z polymer levels. Fifty-eight of 251 had clinically evident portal hypertension (CEPH) at enrollment, and 10 developed CEPH during follow-up. Higher Z AAT polymer levels were associated with existing CEPH (P = .01). In infants without CEPH, higher polymer levels were associated with future CEPH later in childhood, but total AAT was not predictive. Higher gamma-glutamyl transferase (GGT) in the first few months of life was also significantly associated with future CEPH, and risk-threshold GGT levels can be identified. A model was constructed to identify subjects at high risk of future CEPH by combining clinical GGT and polymer levels (area under the curve of 0.83; 95% confidence interval: 0.656–1.00, P = .019). Conclusion: High circulating Z polymer levels and high GGT early in life are associated with future CEPH in AATD, and the use of predictive cutoffs may assist in future clinical trial design

Activation of ULK1 is required for norUDCA-induced AMPK activation and reduction of polymers of α1ATZ.

<p><b>A.</b> Western blotting analysis for phosphorylation of ULK1 (Ser555) in HTOZ cells treated with norUDCA at different concentrations for 1 hour. The lower panel is the densitometry of phospho-ULK1 Ser555 after normalization with total ULK1. Data is expressed as mean ± SD, #<0.05 vs untreated cells. AICAR is used as positive control. <b>B.</b> Western blotting analysis for phosphorylation of ULK1 Ser317, Ser757 and Ser777 in HTOZ cells treated with norUDCA at 200 μM for 1 hour. The left panels are the densitometry of p-ULK1 after normalization with total ULK1, #<0.05 vs untreated cells. <b>C</b>. Western blotting analysis for phosphorylation of ULK1 S555 in HTOZ cells pretreated with compound C (10 μM) for 1 hour and followed by absence or presence of norUDCA at 200 μM for an additional 1 hour. The lower panel is the densitometry of p-ULK1 Ser555 after normalization with total ULK1. Data is expressed as mean ± SD, #<0.05 vs untreated cells and §<0.05 vs norUDCA alone treated cells. Compound C is used as negative control. For A, B and C, total ULK1 is used for normalization and GAPDH is used as a loading control. <b>D</b>. Polymers and monomers of α1ATZ were determined by western blotting analysis in HTOZ cells pretreated with SBI-0206965 (10mM), inhibitor of ULK1, for 1 hour prior to addition of norUDCA at 200 μM for additional 24 hours. The lower panel is the densitometry of α1ATZ after normalization with GAPDH. Data is expressed as mean ± SD, #<0.05 vs untreated cells, §<0.05 vs norUDCA alone treated cells. GAPDH is used as loading control and for normalization.</p