The phenomenal increase in our basic understanding of the molecular mechanism for metal storage diseases (iron and copper) are a paradigm for the future practice of medicine. In each case, the identification of the gene responsible for the disease coupled with our ability to identify patients at risk with mutations are prime examples of how recent advances in biology are altering our clinical management. It is fitting that these two diseases are considered together, as each are autosomal recessive and occur when both copies (alleles) are mutated. Loss of function leads to major pathological consequence, which if untreated causes multiple organ dysfunction including chronic liver disease, cirrhosis and hepatocellular carcinoma. Both diseases share a similar pathophysiological mechanism of excess accumulation of metal at intracellular sites leading to generation of oxygen free radicals and subsequent injury. Finally, the ability to identify individuals at risk for disease by their genetic makeup, referred to as genotype, makes it possible to institute preventative measures to reduce the accumulation of these toxic metals and thereby prevent the development of cellular injury at their target sites, including the liver. Recent advances in molecular diagnosis of these two diseases will be highlighted.

Hemochromatosis, a disease of excess iron storage, is caused by multiple factors including hematological abnormalities requiring transfusion support such as thalassemia or chronic hemolytic anemia, excessive dietary exposure or in association with chronic liver disease such as viral hepatitis or alcoholic liver disease. A distinct subset of iron storage disease, referred to as hereditary hemochromatosis (HH), exists within families, especially those with Northern European ancestry of Nordic or Celtic origin. Estimated carrier rates of 6 to 8% have been reported in this population, making this the most common hereditary disorder in this group. This high carrier rate coupled with ease of preventive treatment by phlebotomy makes this an ideal disease to screen for in this high-risk population.

Clinically, hemochromatosis only presents after significant injury to the target organs, which include the parathyroid, heart, pancreas, liver and pituitary gland. Common symptoms include fatigue, weakness, arthritis and impotence associated with physical findings of congestive heart failure, hepatomegaly, bronze skin and hypogonadism along with cryptogenic "silent" cirrhosis. Patients now rarely present with these so-called "late" findings and most often are detected by increased iron stores as evidenced by increase in transferrin saturation, total iron binding capacity and serum ferritin levels. Previously, the so-called "gold standard" for hemochromatosis was measurement of iron concentration in a liver biopsy sample, which has the added advantage of assessing severity of hepatic injury.

The need to identify patients at risk and to develop a screening test for this disease prompted a search for the gene causing the disease. Association of HLA-A3 on chromosome 6p as a highly predictive marker for HH led to the breakthrough discovery of the hemochromatosis gene in August 1996, known as the HFE gene. HFE shares significant amino acid sequence similarity with other members of the nonpeptide binding HLA receptor gene family suggesting a novel role for this class of proteins in iron regulation. More important, screening of patients with clinical features of HH lead to the remarkable finding of consistent homozygous substitution of the amino acid tyrosine (Y) for cysteine (C) at amino acid position 282, referred to as C282Y. Another mutation, asparate to histidine at position 63 (H63D) was also commonly found (15%) and a compound heterozygote, (C282Y) and (H63D), was found in some rare cases of hemochromatosis.

Substitution of cysteine at position 282 prevents the formation of a critical cysteine-cysteine disulfide bond, which is essential for the proper folding and structure of the HFE protein. The C282Y HFE protein in cell culture is unable to reach the membrane surface, where it can associate with Beta-2 microglobulin (b2m). The HFE - b2m complex is postulated to regulate the activity of the transferrin receptor, which is one of the major pathways for the uptake of cellular iron. Disruption of this HFE - b2m complex is thought to be responsible for excess iron storage by increased activity of the transferrin receptor. Unlike other metals, there are no specific excretory routes for iron other than by loss due to desquamation of cells in the GI tract, skin and urogenital system. The recent finding that the duodenal iron transporter gene is upregulated in patients with homozygous C282Y mutation despite increased total body iron stores suggests that this disregulation may be ultimately responsible for excess iron absorption, which is a hallmark of HH. Persistent imbalance between uptake and elimination of iron eventually overwhelms the cell's capacity to store iron in a protein-bound form.

Clinically, the identification of a common C282Y mutation among a significant number of HH patients of Northern European background has expanded the role of genotyping to a common disease and raises a host of medical, ethical and social issues. Unlike other genetic diseases, the association of this form of hemochromatosis with only two major mutations at this time significantly simplifies the screening technology allowing for commercial testing for these mutations.

Who should be tested and if genotyping should be used for population-based screening remains controversial. At this time, genotyping is not recommended for screening as cost, reproducibility and the existence of other genes responsible for less common forms of HH make this an incomplete screening test. However, genotyping for an individual who presents with evidence of iron overload in the absence of other known causes can be used to confirm the diagnosis, eliminate the need for liver biopsy to formally make the diagnosis and can be helpful in screening first-degree relatives (parents, siblings and children) who need to be identified for institution of preventive phlebotomy. Genotyping may be substituted for liver biopsy to make the formal diagnosis in patients who present with increased iron storage on serum tests (transferrin saturation greater than 45% and serum ferritin greater than 200 but less than 1,000) without hepatomegaly or history of chronic liver disease. Liver biopsy is still indicated to assess liver histology in patients with ferritin greater than 1,000, history of viral hepatitis or alcoholic liver disease, age greater than 40 and to eliminate any unsuspected liver disease. The identification of a homozygous C282Y in these patients confirms the diagnosis of HH and allows one to use genotyping to screen family members to identify individuals at risk. Those found to be homozygous without evidence for iron overloading are candidates for prophylactic phlebotomy or close monitoring of iron storage status. Spouses should be screened for the mutations to assess if children are at risk for being homozygous for the C282Y mutation. Recently, studies have found that C282Y homozygous may not present with evidence for iron overload, indicating that other factors such as environment, genetics or a combination of the two are responsible for varying penetrance of the disease.

The identification of the HFE and its association with b2m has lead to significant advances in how cells handle this essential, yet potentially highly toxic, molecule. The identification of other components in the normal cellular uptake and transport of iron should rapidly add to our understanding of how these proteins modulate iron transport within and among cells. The findings of a common mutation (C282Y) in HH among Northern Europeans suggest that the mutation evolved from a common ancestor who lived approximately 700 years ago and would probably have benefited from increased capacity to absorb iron. In other ethnic groups, the incidence of C282Y mutations in clinical hemochromatosis are much lower, indicating that a non-HFE gene is responsible for iron overload, which might still act on the HFE gene. The identity of these genes and their function in iron transport will add to our understanding of how iron is transported by cells and its disregulation in common liver diseases.