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

Keratin variants associate with progression of fibrosis during chronic hepatitis C infection

Keratins 8 and 18 (K8/K18) protect the liver from various forms of injury. Studies of liver explants from a large cohort of U.S. patients showed that K8/K18 mutations confer a risk to developing end-stage liver diseases, though which diseases are preferentially involved is unknown. We tested the hypothesis that K8/K18 variants are associated with chronic hepatitis C (CHC) and that their presence correlates with progression of fibrosis. Genomic DNA was isolated from peripheral blood of a well-characterized German cohort of 329 patients with CHC infection. Exonic regions were PCR-amplified and analyzed using denaturing high-performance liquid chromatography and DNA sequencing. Our findings showed: (1) amino acid altering keratin heterozygous variants in 24 of 329 CHC patients (7.3%) and non-coding heterozygous variants in 26 patients (7.8%), and (2) 3 new exonic K8 variants (T26R/G55A/A359T); 6 novel non-coding variants and one K18 coding variant (K18 S230T; 2 patients). The most common variants were K8 R341H (10 patients), K8 G62C (6 patients) and K8 I63V (4 patients). A novel and exclusive association of an intronic KRT8 IVS7+10delC deletion in all 10 patients with K8 R341H was observed. Notably, there was a significant association of exonic, but not of intronic K8 variants with increased fibrosis. In conclusion, previously described and novel K8 variants are present in a German population and collectively associate with progression of fibrosis in CHC infection. The unique 100% segregation of the most common K8 variant, R341H, with an intronic deletion suggests that one of these two genetic changes might lead to the other

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

Patient Demographics and Biochemical Values.

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