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The rate of disappearance of intracellular α-l-antitrypsin correlates with liver disease severity in iPScderived hepatocytes generated from PIZZ α-1-antitrypsin deficiency patients

Edgar N. Tafaleng1, 2, Bing Han1, 2, Pamela D. Hale2, 3, Souvik Chakraborty2, 3, Alejandro Soto-Gutierrez2, 4, Carol Feghali-Bostwick5, Darrell Kotton6, Masaki Nagaya1, Stephen A. Duncan7, Donna B. Stolz8, Stephen Strom9, Jayanta Roy-Chowdhury10, David H. Perlmutter2, 3, Ira J. Fox1, 11
1Department of Surgery,University of Pittsburgh School of Medicine, Pittsburgh, PA; 2Children's Hospital of Piffsburgn of UPMC, Pitstburgh, PA; 3Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA; 4Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA; 5Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA; 6Department of Medicine, Boston University School of Medicine, Boston, MA; 7Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI; 8Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA; 9Department of Laboratory Medicine, Division of Pathology, Karolinska Institutet, Stockholm, Sweden; 10Department of Medicine, Albert Einstein College of Medicine, New York, NY; 11 McGowan Institute for Regenerative Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA

Classical α-1-antitrypsin deficiency (ATD) is the most common cause of hereditary pediatric liver disease. The disorder results from the PIZZ mutation that alters the structure of a-1-antitrypsin (AT) and renders it aggregation-prone. This leads to mutant AT accumulation within hepatocytes, causing hepatic injury through a toxic gain-of-function mechanism. Although the PIZZ genotype is necessary for the development of ATD-mediated liver disease, there is variability in liver disease severity among PIZZ individuals. The prevailing hypothesis is that other yet to be identified genetic factors predispose PIZZ individuals to liver disease. Induced pluripotent stem cells (iPScs) from ATD patients with known clinical features could provide an unlimited source of hepatocytes for identifying these modifier genes. We generated 15 iPScs from PIZZ ATD patients with liver disease of varying severity. We differentiated an iPSc line from a severe liver disease ATD patient (severe LD), 3 iPSc clones from mild liver disease ATD patients (mild LD), and a wild-type control iPSc line into hepatocytes. Analysis of severe LD iPSc-derived hepatocytes by electron and confocal microscopy showed poorly organized and dilated rER and intense AT staining localized in the ER and Golgi similar to results seen in severe LD ATD 1° hepatocytes indicating that the cells model the disease. Similar to severe LD ATD 1° hepatocytes, severe LD iPSc-derived hepatocytes also have constitutively activated autophagy as measured by western blot of basal p62 and LC3I-II levels. Using pulse-chase analysis, we detected a slower rate of disappearance of intracellular AT in ATD iPSc-derived hepatocytes (t1/2 = 2.1 to 4 hr) compared to control cells (t1/2≈1.3 hr). More importantly, we found that the delayed disappearance of intracellular AT observed in ATD iPSc-derived hepatocytes is more apparent in cells obtained from the patient with severe LD (t1/2≈ 4 hr) compared to those from the patient with mild LD (t1/2≈ 2.1 hr). This result is consistent with results obtained from severe LD ATD 1° hepatocytes (t1/2≈ 4.2 hr). There was no significant difference between the rates of the two iPSc clones from the mild LD patient, suggesting that the results are not an artifact of reprogramming or differentiation. These results suggest that variation in the severity of liver disease is related to genetic modifiers that affect intracellular degradation pathways.

Disclosures:

Stephen A. Duncan - Consulting: Primorigen Biosciences

Stephen Strom - Stock Shareholder: Stemnion, Yecuris

Ira J. Fox - Advisory Committees or Review Panels: Regenerative Medical Solutions

The following people have nothing to disclose: Edgar N. Tafaleng, Bing Han, Pamela D. Hale, Souvik Chakraborty, Alejandro Soto-Gutierrez, Carol FeghaliBostwick, Darrell Kotton, Masaki Nagaya, Donna B. Stolz, Jayanta Roy-Chowdhury, David H. Perlmutter

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Administration of an iron-deficient diet attenuates dietinduced hepatic steatosis in HFE-associated non-alcoholic fatty liver disease using Hfe-/- mice

Ashley S. Wilkinson1, 2, Kim Bridle1, 2, Laurence Britton1, 2, Lesley Jaskowski1, 2, Linda M. Fletcher3, V Nathan Subramaniam4, 2, Darrell H. Crawford1, 2
1 School of Medicine, The University of Queensland, Greenslopes, QLD, Australia; 2Gallipoli Medical Research Foundation, Greenslopes, QLD, Australia; 3Gastroenterology and Hepatology, Princess Alexandra Hospital, Woolloongabba, QLD, Australia; 4Queensland Institute of Medical Research, Herston, QLD, Australia

Iron and/or HFEmutations have been proposed as having an important role in the progression of injury in subjects with nonalcoholic fatty liver disease (NAFLD). Previous work in our laboratory has shown a greater severity of injury in Hfe -/-mice fed a high calorie diet (HCD) compared with wild-type mice. We examined the contribution of iron to the development of steatosis in an animal model of hemochromatosis by feeding mice an iron deficient diet followed by exposure to a HCD. Methods: Hfe-/- mice were fed either a control diet, HCD, iron-deficient control diet or iron-deficient HCD for 8 weeks (n = 10 per group). Livers were analysed for their hepatic iron concentration (HIC) and quantitative expression of iron metabolism genes by real-time PCR. Histological parameters were staged and graded by a specialist liver pathologist in a blinded fashion. Results: Visceral adipose tissue (VAT) weight was increased in Hfe -/-mice fed HCD compared to the control diet (2.2 ± 0.15 vs 1.2 ± 0.19 g). Liver weights were also increased in Hfe-/ mice fed HCD compared to the control diet. Interestingly, mice fed the iron-deficient HCD had significantly lower liver weights compared to the mice fed HCD (1.26 ± 0.08 vs 1.72 ± 0.11 g). There was a 2.67 fold decrease in hepcidin and a 2.72 fold decrease in ferroportin expression in the livers of mice fed the iron deficient diet compared to the control diet (P< 0.05). Similarly there was a significant decrease in the mice fed the iron deficient HCD compared to the mice fed the HCD. There was also a significant decrease in hepatic iron concentration in the livers of mice fed the iron deficient control diet compared with the control diet (12.5 vs 33.3 μM Fe/g dry weight). Lobular inflammation was decreased in iron-depleted mice fed the control diet and steatosis was decreased in iron-depleted mice fed the HCD (Table 1). Discussion: Our data suggests that in patients with HFEhemochromatosis, it is the increased hepatic iron overload rather than a lack of functional HFEgene itself that potentiates the co-toxic liver injury from a high calorie diet. This work will guide the design of future therapeutic studies for this common clinical problem of elevated iron indices, HFE mutations and NAFLD.

Table 1. Liver Histology Scores.
Histological ParametersControl Diet (nIron Deficient Control Diet (n=10)HCD (n = 10)Iron Deficient HCD (n=10)
  1. Values are expressed as Median (Range).

Steatosis Grade0.5 (0-1)0 (0 - 0)1.5 (0-2)0.5 (0 - 2)
(%) Total Steatosis2.5(0-10)0 (0 - 0)41.5(4 - 60)7.5 (0 - 50)
Lobular Score1(0-2)0(0-1)1(0-1)1(0 −1)
Ballooning0 (0 - 0)0 (0 - 0)0.5 (0-1)0 (0 −1)
NAS Score2 (0 - 3)0(0-1)3(1-4)1.5 (0-2)

Disclosures:

Darrell H. Crawford - Advisory Committees or Review Panels: Roche Products Pty Ltd, Bristol Myers Squibb, Gilead Sciences, Novartis, MSD, Abbvie; Consulting: Roche Products Pty Ltd; Grant/Research Support: Roche Products Pty Ltd; Speaking and Teaching: Roche Products Pty Ltd, Bristol Myers Squibb, Gilead Sciences,MSD

The following people have nothing to disclose: Ashley S. Wilkinson, Kim Bridle, Laurence Britton, Lesley Jaskowski, Linda M. Fletcher, V Nathan Subramaniam

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Iron activation of hepcidin in hemojuvelin knockout mice preferentially targets splenic but not intestinal ferroportin

Konstantinos Gkouvatsos2, Carine Fillebeen2, John Wagner2, Alina Daba2, Giada Sebastiani1, Kostas Pantopoulos2
1Department of Medicine, McGill University Health Centre, Montreal, QC, Canada; 2 Department of Medicine, Lady Davis Institute for Medical Research, McGill University, Montreal, QC, Canada

Hemojuvelin (Hjv) is a bone morphogenetic protein (BMP) coreceptor involved in the regulation of systemic iron homeostasis. Its functional inactivation causes severe iron overload due to suppression of the iron-regulatory hormone hepcidin. To characterize the role of Hjv in iron-sensing, Hjv-/- mice in pure C57BL/6 background were subjected to dietary iron manipulations. Transferrin (Tf) was highly saturated regardless of the dietary iron content, while liver iron deposition was proportional to it. Surprisingly, hepcidin mRNA expression responded to dietary iron intake. Qualitatively, iron-dependent regulation of hepcidin in Hjv-/- mice was similar to isogenic wild type controls, but more than an order of magnitude lower. Iron signaling via the BMP/Smad pathway was preserved but substantially attenuated. The intestinal iron transporters DMT1 and ferroportin were overexpressed. While DMT1 was highly regulated by iron, ferroportin responded to iron only in the spleen but not the duodenum. Our data demonstrate that Hjv is dispensable for iron sensing and solely acts as an enhancer for hepcidin expression. In addition, they suggest a crucial contribution of duodenal DMT1 in iron overload under conditions of Hjv deficiency, and indicate the existence of tissue-specific mechanisms for regulation of ferroportin.

Disclosures:

The following people have nothing to disclose: Konstantinos Gkouvatsos, Carine Fillebeen, John Wagner, Alina Daba, Giada Sebastiani, Kostas Pantopoulos

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Open label, phase-II clinical study, to evaluate the efficacy of lanreotide 90mg in symptomatic polycystic liver disease, including dose escalation at month 6 in nonresponders

Frederik J. Temmerman1, Thien Anh Ho2, Ragna Vanslembrouck3, Walter Coudyzer3, Vincent De Ruyter4, Jos van Pelt1, Bert Bammens5, Yves Pirson2, Frederik Nevens1
1Department and Laboratory of Hepatology, Leuven, University Hospitals, KULeuven, Leuven, Belgium; 2Department of Nephrology, Brussels, Université Catholique de Louvain, Brussels, Belgium; 3Department of Radiology, Leuven, University Hospitals, KULeuven, Leuven, Belgium; 4Medical Advisor, IPSEN, Merelbeke, Belgium; 5Department of Nephrology, Leuven, University Hospitals, KULeuven, Leuven, Belgium

BACKGROUND: The somatostatin analogue Lanreotide autogel (LAN) 120mg reduces liver volume (LV) in patients (pts) with polycystic liver disease (PCLD). Steatorrhea and abdominal cramps are well known side-effects. The efficacy of lower LAN dose and the susceptibilty to tachyphylaxis of LAN in PCLD is not known. AIM: To investigate the efficacy & long-term effect of 90mg in PCLD and whether dose escalation is meaningful in non-responders. METHODS: All pts received LAN 90mg/4 weeks. In case of change in LV<100mL (non responder) at month 6, dose was increased to 120mg/4 wks; responders remained on 90mg/4wks. The total study duration was 18 months. Primary endpoint was the change in LV from baseline at 6, 12, 18 months (CT scan). Secondary end point was the effect on kidney volumes. The level of significance was 0.05 (NCT01315795). RESULTS: 59 pts (women/men: 52/7; 51y) were included; 53 reached the 6 month evaluation. 16/53 (30%) were responders reaching the 18 month endpoint: the absolute change in LV was −395mL (IQR: −732; −221). 23/53 needing dose escalation reached 18 months. The relative changes in both groups over 18 months are presented in the graph below. Throughout the study, 20 pts dropped out: underwent LT (n=7), experienced severe side-effects: steatorrhea (n=6) / severe abdominal cramps (n=1), or other reasons (n=6). Pts with a decrease >100mL after 6 months tended to have larger LV's at baseline (5960mL (IQR: 3973; 7836) compared to those not reaching this cutt-off (4948mL (IQR: 3724; 5790) (MannWhitney test; two-tailed; p=0.1). 30/39 pts ending 18 months had renal cystic disease: kidney volumes (responders n=13; nonresponders n=17) showed no significant changes (One Way ANOVA on ranks, resp.; p=0.3; p=0.13). CONCLUSION: LAN 90mg is effective in decreasing LV in PCLD especially in pts with larger LV's; and this was maintained 1.5 year. Dose escalation to LAN 120mg in case of non-responders was effective.

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Disclosures:

Vincent De Ruyter - Employment: Ipsen NV

Bert Bammens - Consulting: Amgen; Grant/Research Support: Baxter Healthcare, Roche, Sanofi-Aventis, Astellas, Amgen; Speaking and Teaching: Baxter Healthcare

Frederik Nevens - Grant/Research Support: Ipsen, Roche, MSD, Astellas, CAF

The following people have nothing to disclose: Frederik J. Temmerman, Thien Anh Ho, Ragna Vanslembrouck, Walter Coudyzer, Jos van Pelt, Yves Pirson

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Relapse of porphyria cutanea tarda after achieving remission with phlebotomy or low dose hydroxychloroquine

Ashwani K. Singal1, 4, Eric Gou2, Marisol Albuerne3, Csilla Kormos Hallberg2, Karl E. Anderson2,4
1 Gastroenterliogy and Hepatliogy, UAB, Birmingham, AL; 2Preventive Medicine and Community Health, UTMB, Galveston, TX; 3Internal Medicine, UTMB, Gaiveston, TX; 4Division of Gastroenterology and Hepatology, UTMB, Galveston, TX

Background: Porphyria cutanea tarda (PCT) is due to inhibition of hepatic uroporphyrinogen decarboxylase (UROD) activity and is readily treated by phlebotomy or oral low-dose hydroxychloroquine (HCQ, 100 mg twice weekly), which our recent findings suggest are equally effective and safe. Data comparing relapse of PCT after these treatments are scanty. Methods and Results: Thirty-nine patients seen at one site achieved remission (defined as a normal plasma porphyrin concentration of ≤0.9 mcg/dL, with cessation of new skin lesions) and were followed for relapse of PCT. Median follow up was 1 year. Thirty of these 39 patients had participated in a prospective study (Clin Gastroenterol Hepatol 2012, 10, 1402-1409) comparing treatment by phlebotomy (N = 17, endpoint: serum ferritin of <20 ng/L, followed by plasma porphyrins ≤0.9 mcg/dL) or low-dose HCQ (N = 13, endpoint: plasma porphyrins <0.9 mcg/dL for at least one month). Fifteen of 39 (39%) patients relapsed biochemically (29% of 24 after phlebotomy and 53% of 15 after HCQ; Log Rank P=0.14). Relapse rates at 1, 3, and 5 years after phlebotomy or HCQ were 8% vs. 25%, 29% vs. 33%, and 47% vs. 53% respectively. Relapse rates after these treatments did not differ by age, sex, race, alcohol use, smoking, chronic hepatitis C, homozygous or double heterozygous hemochromatosis gene (HFE) mutations, estrogen use, baseline serum ferritin, or time to remission, but numbers for these comparisons were small. One of 24 non-relapsing patients and 2 of 15 relapsing patients had heterozygous UROD mutations. Relapsing patients had higher pretreatment plasma porphyrin concentrations compared to non-relapsers (16±21 vs. 5 ±5 mcg/dL; P=0.03). All remained abstinent from alcohol during treatment. Data were available on resumption of alcohol use in 30 patients, and this was not more common in relapsers than non-relapsers (9 of 15 vs. 10 of 15, respectively (P=0.95). HCV treatment was given to 3 of 15 patients in each group (P=0.99). Conclusions: PCT relapses may be more frequent with more severe disease and after achieving remission with low-dose HCQ than phlebotomy. Longer follow-up and a larger sample size are planned with the Porphyrias Consortium Longitudinal Study. Grant support from the National Institutes of Health for the Porphyrias Consortium (1 U54 DK083909) of the NIH Rare Diseases Clinical Research Network and a Clinical and Translational Science Award (UL1TR000071) and the American Porphyria Foundation.

Disclosures:

The following people have nothing to disclose: Ashwani K. Singal, Eric Gou, Marisol Albuerne, Csilla Kormos Hallberg, Karl E. Anderson

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Wilson's disease: effects of gestational methyl group supplementation on global DNA methylation and gene expression in fetal mouse liver

Valentina Medici1, Noreene Shibata1, Kusum K. Kharbanda2, Mohammad S. Islam3, Charles H. Halsted1, Janine M. LaSalle3
1Internal Medicine, University of California Davis, Sacramento, CA; 2Research Service, Veterans Affairs Nebraska-Western lowa Health Care System, Omaha, NE; 3Medical Microbiology and Immunology, University of California Davis, Davis, CA

Background & Aims. Although numerous mutations are known to impair copper transport function in Wilson's disease (WD), the failure to identify specific genotype-to-phenotype correlations could be due to environmental, nutritional, and epigenetic factors that influence lifelong phenotype expression. Previously we showed that methionine metabolism, which is central in the regulation of methylation reactions, is altered in WD and may regulate the expression of genes involved in liver damage. Since maternal diet is known to affect fetal gene expression through epigenetic mechanisms, we propose the heredity of WD may be influenced by maternal methylation status. Methods. The tx-j mouse model of WD and wildtype C3H female mice were started on choline-supplemented (choline 36 mmol/Kg of diet) and control diets (choline 8 mmol/Kg of diet) 2 weeks before mating and through pregnancy to embryonic day 17.Livers of 6-11 embryos/dam were collected for study of liver copper levels; transcript levels of selected genes related to cell cycle; methionine metabolism enzymes and hepatic steatosis by qPCR; S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) hepatic levels by HPLC; and hepatic global DNA methylation by dot blot analysis. Results. Compared to fetal livers from control mice, livers from tx-j mice presented reduced copper levels (128±11 vs 24±7.4 nmol/g, p< 0.01) most likely related to lack of ceruloplasmin in WD dams with consequent reduced copper transport to the fetus. Tx-j fetal livers had 1.5 to 100 times down-regulation of transcript levels of cyclin D1 and of genes related to methionine metabolism (including methionine synthase, methionine adenosyltransferase2A, DNA methyltransferases, and S-adenosylhomocysteine hydrolase) (p<0.05). A similar down-regulation was observed in transcript levels related to lipid metabolism including sterol regulatory element-binding protein-1 c and peroxisome proliferator-activated receptor alpha (p<0.05 for each). Gene transcript down-regulation was corrected to control levels by choline supplementation (wildtype untreated vs tx-j cholinesupplemented, p=n. s.). Whereas there was no change in SAM or SAH levels after choline supplementation, global DNA methylation increased by 17% in response to choline treatment in tx-j mice (p<0.05). Conclusions. Supplementation with methyl group donor choline during pre-conception and fetal life in the tx-j mouse model of WD can increase liver gene transcripts to control levels in fetal livers of mice model of WD by mechanisms that may be related to cell growth. Our results suggest that the WD phenotype can potentially be modified by maternal in utero factors.

Disclosures:

The following people have nothing to disclose: Valentina Medici, Noreene Shibata, Kusum K. Kharbanda, Mohammad S. Islam, Charles H. Halsted, Janine M. LaSalle