Non-Coding Keratin Variants Associate with Liver Fibrosis Progression in Patients with Hemochromatosis

Background: Keratins 8 and 18 (K8/K18) are intermediate filament proteins that protect the liver from various forms of injury. Exonic K8/K18 variants associate with adverse outcome in acute liver failure and with liver fibrosis progression in patients with chronic hepatitis C infection or primary biliary cirrhosis. Given the association of K8/K18 variants with endstage liver disease and progression in several chronic liver disorders, we studied the importance of keratin variants in patients with hemochromatosis. Methods: The entire K8/K18 exonic regions were analyzed in 162 hemochromatosis patients carrying homozygous C282Y HFE (hemochromatosis gene) mutations. 234 liver-healthy subjects were used as controls. Exonic regions were PCRamplified and analyzed using denaturing high-performance liquid chromatography and DNA sequencing. Previouslygenerated transgenic mice overexpressing K8 G62C were studied for their susceptibility to iron overload. Susceptibility to iron toxicity of primary hepatocytes that express K8 wild-type and G62C was also assessed. Results: We identified amino-acid-altering keratin heterozygous variants in 10 of 162 hemochromatosis patients (6.2%) and non-coding heterozygous variants in 6 additional patients (3.7%). Two novel K8 variants (Q169E/R275W) were found. K8 R341H was the most common amino-acid altering variant (4 patients), and exclusively associated with an intronic KRT8 IVS7+10delC deletion. Intronic, but not amino-acid-altering variants associated with the development of liver fibrosis. I

Serum/Plasma Proteome in Non-Malignant Liver Disease

The liver is the central metabolic organ and produces 85–90% of the proteins found in plasma. Accordingly, the plasma proteome is an attractive source of liver disease biomarkers that reflects the different cell types present in this organ, as well as the processes such as responses to acute and chronic injury or the formation of an extracellular matrix. In the first part, we summarize the biomarkers routinely used in clinical evaluations and their biological relevance in the different stages of non-malignant liver disease. Later, we describe the current proteomic approaches, including mass spectrometry and affinity-based techniques, that allow a more comprehensive assessment of the liver function but also require complex data processing. The many approaches of analysis and interpretation and their potential caveats are delineated. While these advances hold the promise to transform our understanding of liver diseases and support the development and validation of new liver-related drugs, an interdisciplinary collaboration is needed

Desertification indicators as diagnosis criteria for desertification risk assessment in Europe

8 páginas, 4 figuras.Despite the seriousness of the environmental and socio-economic impacts of desertification, few efforts have been made to devise diagnostic and monitoring techniques for appraising the status and trend of desertification. Indicators are integrated and synthetic information that can provide data on threshold levels, status and evolution of relevant physical, chemical, biological and anthropogenic processes. Existing studies have taken a global perspective, and most have dealt with rangeland conditions. Moreover, the zones usually studied have included countries whose socio-economic and cultural backgrounds differ from those prevailing in Europe. This paper presents a methodology and discussion of indicator requirements, selection and definition, procedures for measuring or making estimates, sensitivity, range of values and interactions. Emphasizing the need to use indicators to develop a system of desertification evaluation, this paper outlines the advantages and difficulties of using such an evaluation system.We would like to thank BANCAIXA for its financial support of this study.Peer reviewe

Distribution of Exonic Keratin Variants.

<p>The table displays the number of patients with and without liver cirrhosis for the listed keratin variants.</p>*<p>The highlighted variant is considered to be a “polymorphism” rather than a “mutation” that is likely to have biologic significance.</p>†<p>Novel variants which were not previously described.</p>$<p>One female patient harbored 2 independent amino acid altering KRT8 variants (R341H+A319S). N.T.not tested.</p

Distribution of Non-Coding Keratin Variants.

<p>The table displays the number of patients with and without liver cirrhosis for the listed keratin variants.</p>*<p>IVS7+10delC variant completely associates with KRT8 R341H variant and is not included in the count of total intronic variants.</p>†<p>The highlighted variant was found only in one control subject (out of 234).</p>**<p><b><i>p</i></b><b> = 0.02</b>.</p

The K8 G62C variant does not influence the extent of hepatocellular iron accumulation.

<p>To test, whether hepatocellular iron accumulation is affected by the presence of keratin variants, transgenic mice overexpressing wild-type human keratin 8 (K8 WT) or K8 G62C variant were fed with 3% carbonyl iron-containing diet for one month (Iron) and compared to age-matched animals kept on standard diet (Control) (n = 4 mice/group). Iron-feeding did not lead to an increase in serum ALT (<b>A</b>), but caused a significant elevation in hepatic iron concentration (HIC) (<b>B</b>). There was no significant difference in ALT or HIC levels between K8 WT and G62C mice after iron feeding. (<b>C</b>) Perl's Prussian blue staining revealed a similar pattern of iron deposition in both mouse lines. Scale bar = 200 µm.</p

Patient Demographics and Biochemical Values.

<p>Abbreviations: LBx:liver biopsy; SD: standard deviation; HIC: hepatic iron concentration; HII:hepatic iron index.</p

The K8 G62C variant does not affect iron toxicity in ex vivo cultured hepatocytes.

<p>To study the hepatocellular iron toxicity, primary hepatocytes from transgenic animals overexpressing K8 WT or K8 G62C were cultured in normal media (Control) or media supplemented with 100 µM nitrilotriacetic acid (NTA) or 100 µM iron-NTA (FeNTA) for 24 hours. FeNTA, but not NTA treatment led to a significant increase in LDH (<b>A</b>) and ALT levels (<b>C</b>) in cell culture supernatants together with significant decrease in cell viability as assessed by MTT assay (<b>B</b>). The enzyme levels and cell viability did not differ significantly between K8 WT and K8 G62C hepatocytes.</p

The Clinical Features of Hemochromatosis Patients Harboring Exonic and Intronic Keratin 8 Variants.

<p>Abbreviations: LBx:liver biopsy; SD: standard deviation; HIC: hepatic iron concentration; HII: hepatic iron index.</p><p>Note that intronic keratin variants were preferentially found in male subjects (<i>p = 0.09</i> for distribution among the sexes).</p