Examination of HFE C282Y/H63D Heterozygotes as a Potential Human Modeling System for Low Level Liver Damage

摘要

Individuals heterozygous for hemochromatosis gene (HFE) mutations have been shown to demonstrate mild systemic iron-loading. In some studies, mildly elevated liver enzymes are noted. More sensitive testing methodologies are needed to assist in determining which heterozygous individuals may be at risk for future deleterious effects. Alpha Glutathione-S-Transferase (AGST) has been used to determine sub-clinical liver dysfunction and damage. However, AGST has not been used to examine the potential damage caused by mild iron loading. Whole blood/serum samples from individuals 20-50 years of age were collected from the Wright Patterson AFB clinical laboratory with only age and sex indicated. Genomic DNA was isolated from anticolagulated whole blood using the GFX Genomic Blood DNA Purification Kit (Amersham Biosciences). In two reactions, Exon 2 and Exon 4 of the HFEl gene were amplified using primer sets HH63A/HH63B and HHl/HH5, respectively, in a standard PCR reaction (Accuprime PCR Kit, Invitrogen). The PCR products were digested either with MboI (Exon 2 containing the C186G mutation) or RsaI (Exon 4 containing the G845A mutation). The resultant restriction fragments were analyzed with matched controls on a 2% TBE agarose gel. Three groups of samples were identified (C282Y heterozygotes, H63D heterozygotes, and homozygous normal controls). Serum alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase, alkaline phosphatase, iron status, and AGST were analyzed on each sample. Samples from individuals possessing heterozygous C282Y mutation did not demonstrate a statistically significant elevation in AGST or other liver enzymes when compared to samples with no mutation. Similar results were found in individuals heterozygous for the H63D mutation. These results demonstrate that AGST activity may not be a good indicator for sub-clinical liver damage caused by increased loading of hepatic iron.