Lesley A. Jaskowski

Classifying MLH1 and MSH2 variants using bioinformatic prediction, splicing assays, segregation, and tumor characteristics

Reliable methods for predicting functional consequences of variants in disease genes would be beneficial in the clinical setting. This study was undertaken to predict, and confirm in vitro, splicing aberrations associated with mismatch repair (MMR) variants identified in familial colon cancer patients. Six programs were used to predict the effect of 13 MLH1 and 6 MSH2 gene variants on pre‐mRNA splicing. mRNA from cycloheximide‐treated lymphoblastoid cell lines of variant carriers was screened for splicing aberrations. Tumors of variant carriers were tested for microsatellite instability and MMR protein expression. Variant segregation in families was assessed using Bayes factor causality analysis. Amino acid alterations were examined for evolutionary conservation and physicochemical properties. Splicing aberrations were detected for 10 variants, including a frameshift as a minor cDNA product, and altered ratio of known alternate splice products. Loss of splice sites was well predicted by splice‐site prediction programs SpliceSiteFinder (90%) and NNSPLICE (90%), but consequence of splice site loss was less accurately predicted. No aberrations correlated with ESE predictions for the nine exonic variants studied. Seven of eight missense variants had normal splicing (88%), but only one was a substitution considered neutral from evolutionary/physicochemical analysis. Combined with information from tumor and segregation analysis, and literature review, 16 of 19 variants were considered clinically relevant. Bioinformatic tools for prediction of splicing aberrations need improvement before use without supporting studies to assess variant pathogenicity. Classification of mismatch repair gene variants is assisted by a comprehensive approach that includes in vitro, tumor pathology, clinical, and evolutionary conservation data. Hum Mutat 0, 1–14, 2009.

Excess iron modulates endoplasmic reticulum stress-associated pathways in a mouse model of alcohol and high-fat diet-induced liver injury

Endoplasmic reticulum (ER) stress is an important pathogenic mechanism for alcoholic (ALD) and nonalcoholic fatty liver disease (NAFLD). Iron overload is an important cofactor for liver injury in ALD and NAFLD, but its role in ER stress and associated stress signaling pathways is unclear. To investigate this, we developed a murine model of combined liver injury by co-feeding the mildly iron overloaded, the hemochromatosis gene-null (Hfe−/) mouse ad libitum with ethanol and a high-fat diet (HFD) for 8 weeks. This co-feeding led to profound steatohepatitis, significant fibrosis, and increased apoptosis in the Hfe−/− mice as compared with wild-type (WT) controls. Iron overload also led to induction of unfolded protein response (XBP1 splicing, activation of IRE-1α and PERK, as well as sequestration of GRP78) and ER stress (increased CHOP protein expression) following HFD and ethanol. This is associated with a muted autophagic response including reduced LC3-I expression and impaired conjugation to LC3-II, reduced beclin-1 protein, and failure of induction of autophagy-related proteins (Atg) 3, 5, 7, and 12. As a result of the impaired autophagy, levels of the sequestosome protein p62 were most elevated in the Hfe−/− group co-fed ethanol and HFD. Iron overload reduces the activation of adenosine monophosphate protein kinase associated with ethanol and HFD feeding. We conclude that iron toxicity may modulate hepatic stress signaling pathways by impairing adaptive cellular compensatory mechanisms in alcohol- and obesity-induced liver injury.

The serum hepcidin:ferritin ratio is a potential biomarker for cirrhosis

Serum hepcidin concentration is potentially affected by inflammation and iron stores in chronic liver disease (CLD), but little is known about the relationship between hepcidin and the degree of hepatic fibrosis. We investigated the potential role of serum hepcidin as a biomarker of advanced liver disease.

Altered Lipid Metabolism in Hfe-Knockout Mice Promotes Severe NAFLD and Early Fibrosis

The HFE protein plays a crucial role in the control of cellular iron homeostasis. Steatosis is commonly observed in HFE-related iron-overload disorders, and current evidence suggests a causal link between iron and steatosis. Here, we investigated the potential contribution of HFE mutations to hepatic lipid metabolism and its role in the pathogenesis of nonalcoholic fatty liver disease. Wild-type (WT) and Hfe knockout mice (Hfe(-/-)) were fed either standard chow, a monounsaturated low fat, or a high-fat, high-carbohydrate diet (HFD) and assessed for liver injury, body iron status, and markers of lipid metabolism. Despite hepatic iron concentrations and body weights similar to WT controls, Hfe(-/-) mice fed the HFD developed severe hypoxia-related steatohepatitis, Tnf-α activation, and mitochondrial respiratory complex and antioxidant dysfunction with early fibrogenesis. These features were associated with an upregulation in the expression of genes involved in intracellular lipid synthesis and trafficking, while transcripts for mitochondrial fatty acid β-oxidation and adiponectin signaling-related genes were significantly attenuated. In contrast, HFD-fed WT mice developed bland steatosis only, with no inflammation or fibrosis and no upregulation of lipogenesis-related genes. A HFD led to reduced hepatic iron in Hfe(-/-) mice compared with chow-fed mice, despite higher serum iron, decreased hepcidin expression, and increased duodenal ferroportin mRNA. In conclusion, our results demonstrate that Hfe(-/-) mice show defective hepatic-intestinal iron and lipid signaling, which predispose them toward diet-induced hepatic lipotoxicity, accompanied by an accelerated progression of injury to fibrosis.

Lack of efficacy of mTOR inhibitors and ACE pathway inhibitors as antifibrotic agents in evolving and established fibrosis in Mdr2-/- mice

Mammalian target of rapamycin and angiotensin‐converting enzyme inhibition has been shown to have antifibrotic activity in models of liver fibrosis. The aim of our study was to determine the efficacy of rapamycin, everolimus, irbesartan and captopril, alone and in combination, as antifibrotic agents in the Mdr2−/− model of cholestasis both in early injury and established disease.

Combination curcumin and vitamin E treatment attenuates diet-induced steatosis in Hfe-/- mice

AIM To investigate the synergistic hepato-protective properties of curcumin and vitamin E in an Hfe-/- high calorie diet model of steatohepatitis. METHODS Hfe-/- C57BL/6J mice were fed either a high calorie diet or a high calorie diet with 1 mg/g curcumin; 1.5 mg/g vitamin E; or combination of 1 mg/g curcumin + 1.5 mg/g vitamin E for 20 wk. Serum and liver tissue were collected at the completion of the experiment. Liver histology was graded by a pathologist for steatosis, inflammation and fibrosis. RNA and protein was extracted from liver tissue to examine gene and protein expression associated with fatty acid oxidation, mitochondrial biogenesis and oxidative stress pathways. RESULTS Hfe-/- mice fed the high calorie diet developed steatohepatitis and pericentral fibrosis. Combination treatment with curcumin and vitamin E resulted in a greater reduction of percent steatosis than either vitamin E or curcumin therapy alone. Serum alanine aminotransferase and non-alcoholic fatty liver disease (NAFLD) activity score were decreased following combination therapy with curcumin and vitamin E compared with high calorie diet alone. No changes were observed in inflammatory or fibrosis markers following treatment. Epididymal fat pad weights were significantly reduced following combination therapy, however total body weight and liver weight were unchanged. Combination therapy increased the mRNA expression of AdipoR2, Ppar-α, Cpt1a, Nrf-1 and Tfb2m suggesting enhanced fatty acid oxidation and mitochondrial biogenesis. In addition, combination treatment resulted in increased catalase activity in Hfe-/- mice. CONCLUSION Combination curcumin and vitamin E treatment decreases liver injury in this steatohepatitis model, indicating that combination therapy may be of value in NAFLD.

Inconsistent hepatic antifibrotic effects with the iron chelator deferasirox.

Development of effective antifibrotic treatments that can be translated to clinical practice is an important challenge in contemporary hepatology. A recent report on β‐thalassemia patients demonstrated that deferasirox treatment reversed or stabilized liver fibrosis independent of its iron‐chelating properties. In this study, we investigated deferasirox in cell and animal models to better understand its potential antifibrotic effects.

Low dose lipopolysaccharide causes biliary injury by blood biliary barrier impairment in a rat hepatic ischemia‐reperfusion model

This study explored whether bacterial endotoxins, in the form of lipopolysaccharides (LPS), could have an injurious effect on the biliary tract in conjunction with ischemia. A total of 64 rats were randomly assigned to 4 groups: sham operation (sham group), 1 mg/kg LPS intraperitoneal (LPS group), hepatic ischemia/reperfusion (IR; IR group), and IR combined with LPS (IR+LPS group). Following 1 or 6 hours of reperfusion, serum liver tests, bile duct histology, immunofluorescence microscopy (zonula occludens‐1 [ZO‐1]), bile composition (bile salts, phospholipids, lactate dehydrogenase), hepatic gene expression (bile salt transporters and inflammatory mediators), as well as serum and biliary cytokine concentrations were quantified and compared between the study groups. In addition, the integrity of the blood biliary barrier (BBB) was assayed in vivo using horseradish peroxidase (HRP). LPS administration induced severe small bile duct injury following 6 hours of reperfusion. Furthermore, total bile salts and bilirubin concentrations in serum were increased in the LPS groups compared with sham controls (LPS, + 3.3‐fold and +1.9‐fold; IR+LPS, + 3.8‐fold and +1.7‐fold, respectively). The BBB was impaired in the LPS groups as evidenced by elevated levels of HRP in bile (+4.9‐fold), and decreased expression of claudin 1 (–6.7‐fold) and claudin 3 (–3.6‐fold). LPS was found to be a potent inducer of small bile duct injury following hepatic ischemia and 6 hours of reperfusion. This injury was associated with increased permeability of the BBB and impaired hepatic bile salt clearance. Liver Transplantation 23 194–206 2017 AASLD

Heterozygous Hfe gene deletion leads to impaired glucose homeostasis, but not liver injury in mice fed a high‐calorie diet

Heterozygous mutations of the Hfe gene have been proposed as cofactors in the development and progression of nonalcoholic fatty liver disease (NAFLD). Homozygous Hfe deletion previously has been shown to lead to dysregulated hepatic lipid metabolism and accentuated liver injury in a dietary mouse model of NAFLD. We sought to establish whether heterozygous deletion of Hfe is sufficient to promote liver injury when mice are exposed to a high‐calorie diet (HCD). Eight‐week‐old wild‐type and Hfe+/− mice received 8 weeks of a control diet or HCD. Liver histology and pathways of lipid and iron metabolism were analyzed. Liver histology demonstrated that mice fed a HCD had increased NAFLD activity score (NAS), steatosis, and hepatocyte ballooning. However, liver injury was unaffected by Hfe genotype. Hepatic iron concentration (HIC) was increased in Hfe+/− mice of both dietary groups. HCD resulted in a hepcidin‐independent reduction in HIC. Hfe+/− mice demonstrated raised fasting serum glucose concentrations and HOMA‐IR score, despite unaltered serum adiponectin concentrations. Downstream regulators of hepatic de novo lipogenesis (pAKT, SREBP‐1, Fas, Scd1) and fatty acid oxidation (AdipoR2, Pparα, Cpt1) were largely unaffected by genotype. In summary, heterozygous Hfe gene deletion is associated with impaired iron and glucose metabolism. However, unlike homozygous Hfe deletion, heterozygous gene deletion did not affect lipid metabolism pathways or liver injury in this model.

Heterozygous Deletion of the Hfe Gene Contributes to Hepatic Iron Loading But Not Steatosis in Mice Fed a High Fat Diet

Introduction: The liver is central to the metabolism of both iron and cholesterol. Cholesterol is synthesised and further metabolised to bile acids in the liver and the liver plays an important role in regulation of iron metabolism. It is also the organ in which excess iron is stored. Clinically, links have been noted between lipid and iron metabolism, with approximately one - third of patients with non - alcoholic fatty liver disease exhibiting altered iron parameters. On a molecular level, we have previously reported that wild - type mice fed iron - deficient, normal or iron - loaded diets exhibited increased hepatic cholesterol and increased hepatic gene expression of enzymes in the cholesterol biosynthesis pathway with increasing hepatic iron burden. In the genetic disorder, haemochromatosis, the liver can become overloaded with iron; however, clinical studies have suggested that lipid metabolism may not be perturbed in haemochromatosis. Methods and Materials: We investigated hepatic cholesterol metabolis m in three mouse models of hereditary haemochromatosis: Hfe - / - , Tfr2 Y245X single mutant and Hfe - / - x Tfr2 Y245X double mutant animals as well as wild - type controls. Mice were fed normal mouse chow and sacrificed at 10 weeks of age. Hepatic gene expression, total cholesterol and non – haem iron were measured. Liver non - haem iron was similar in Hfe - / - and Tfr2 Y245X mice (16.6±0.8 and 17±1 μmol Fe /g liver, respectively) and significantly higher in the double mutant animals (22.4±0.7 μmol Fe /g liver ; P<0.004) than either of the single mutant mice. Results: Only one group of genes increased significantly with increasing hepatic iron: those involved in cholesterol transport. Gene expression of apolipoproteins A4, C1, C2, C3 and E increased significantly with increasing hepatic iron as did expression of VLDL receptor. In contrast to our findings in wild - type mice, gene expression of cholesterol biosynthetic enzymes did not increase significantly with liver iron burden and there were no differences in hepatic cholesterol between the groups of mutant mice. We also measured expression of genes involved in cholesterol regulation, which similarly, did not increase with increasing hepatic iron. Approximately 50% of cholesterol synthesised in the liver is directed to bile acid synthesis; however, gene expression of bile acid pathway enzymes did not change with respect to hepatic iron burden. Conclusion: These results suggest that iron - associated cholesterol regulation may be ameliorated by the genetic changes which occur in haemochromatosis.Poster presented at Fifth Congress of the International BioIron Society that took place in University College London (London, United Kingdom) during 14-18th April 2013.