Hemochromatosis: Discovery of the HFE Gene

Abstract

Hereditary hemochromatosis (HH) is a common inherited disorder of iron metabolism affecting about 1 in 250 individuals. HH results in an increased absorption of iron at the baso-lateral surface of the enterocyte with aberrant regulation of ferroportin-mediated transfer of iron in turn brought on by a decrease in circulating hepcidin. The medical literature describes a colorful history of HH with important contributions from faculty at Saint Louis University.

Overview of Hereditary Hemochromatosis

Hereditary hemochromatosis (HH) is a common inherited disorder of iron metabolism which can lead to an increase in systemic iron levels. This is a consequence of excessive intestinal absorption of dietary iron. Most (approximately 85%) patients with HH have mutations in the HFE gene, and HFE-related HH is one of the most common inherited disorders among whites, with a frequency of about 1 in 250.

Two independent mutations of the HFE gene are principally responsible for HFE-related HH. These mutations result in a change of cysteine to tyrosine at amino acid 282 (C282Y) and of histidine to aspartic acid at amino acid 63 (H63D) of the HFE protein. Approximately 95% of persons with HFE-related HH are homozygous for the C282Y mutation. Population studies indicate that the penetrance of the C282Y mutation is incomplete, and genetic modifiers may be involved. Some compound heterozygotes with copies of both the C282Y and H63D mutations have a clinically significant degree of iron overload.

The pathogenesis of nearly all forms of HH involves an inappropriately low expression of the iron-regulatory hormone hepcidin, which acts to decrease the export of iron from absorptive enterocytes and reticuloendothelial cells. Hepcidin is highly expressed in hepatocytes, and it is proposed that HFE protein, TFR2, and hemojuvelin all play a role in the hepatic iron-signalling pathway that regulates hepcidin expression. The C282Y mutation causes functional inactivation of the HFE protein, leading to low hepcidin expression with a resultant increase in duodenal iron absorption (See Figure 1).

Figure 1.

In the duodenal enterocyte, dietary iron is reduced to the ferrous state by duodenal ferric reductase (Dcytb), transported into the cell by divalent metal transporter 1 (DMT1), and released by way of ferroportin into the circulation. Hephaestin facilitates enterocyte iron release. Hepatocytes take up iron from the circulation either as free iron or transferrin-bound iron (through transferrin receptor 1 and transferrin receptor 2). Transferrin receptor 2 may serve as a sensor of circulating transferrin-bound iron, thereby influencing expression of the iron regulatory hormone hepcidin. The hepcidin response is also modulated by HFE and hemojuvelin. Hepcidin is secreted into the circulation, where it down-regulates the ferroportin-mediated release of iron from enterocytes, macrophages, and hepatocytes (dashed red lines).

In HFE-related HH, the excess iron is preferentially deposited in the cytoplasm of parenchymal cells of various organs and tissues, including the liver (See Figure 2) pancreas, heart, endocrine glands, skin, and joints. Damage can result in micronodular cirrhosis of the liver and atrophy of the pancreas (primarily islets). Hepatocellular carcinoma, usually in the presence of cirrhosis, is another consequence of excess iron deposition in the liver. Symptoms are related to damage of the involved organs and include liver failure (from cirrhosis), diabetes mellitus, arthritis, cardiac dysfunction (arrhythmias and heart failure), and hypogonadotropic hypogonadism.

Figure 2.

Low power view of liver biopsy sample from a patient with C282Y homozygous hemochromatosis. Iron stains blue and is found in hepatocytes with a periportal to pericentral gradient (Perls’ Prussian blue, x 100).

The diagnosis of iron overload includes serum iron studies (elevated transferrin saturation, elevated serum ferritin levels), genetic testing, and sometimes liver biopsy to assess the hepatic iron concentration and degree of liver injury. In cases of HFE-related HH, liver biopsy is usually not indicated if the patient has normal liver enzyme levels and a serum ferritin level below 1,000 ng/mL. Because regular phlebotomy therapy prevents or reverses the accumulation of excess iron and prevents the complications of HH, it is important to identify persons with this inherited disorder early in the disease process.¹

History of HH

The first medical description of a patient with hemochromatosis was by Trousseau² in the French pathology literature in 1865. Twenty-four years later, the German pathologist von Recklinghausen³ was the first to use the term hemochromatosis; he thought that the pigmentation (“chrom”) in the tissues of patients with the disorder was caused by something circulating in their blood (“hemo”). In 1935, Joseph Sheldon,⁴ a British geriatrician, published a monograph describing the 311 cases of hemochromatosis that existed in the world literature up to that time. Sheldon concluded that hemochromatosis was an inherited disorder in which tissue injury and damage resulted from excess iron deposition. He drew accurate conclusions without the techniques of modern molecular medicine available today. The situation was somewhat confused by MacDonald,⁵ a pathologist at the Boston City Hospital, who believed that hemochromatosis was a nutritional disorder, possibly because he saw many alcoholic patients who happened to be of Irish descent. It is now known that the prevalence of homozygosity for HFE-related hereditary hemochromatosis (HH) is high in the Irish population, approaching 1 in 70 persons.⁶ In 1976, Marcel Simon and colleagues⁷ definitively showed that classic hemochromatosis is inherited as an autosomal recessive disorder, with linkage to the human leukocyte antigen (HLA) region of the human genome; the gene for hemochromatosis is located on the short arm of chromosome 6. It took another 20 years until the research group at Mercator Genetics successfully identified and cloned the hemochromatosis gene by means of a positional cloning approach using DNA samples from well-documented patients with hemochromatosis in the United States.⁷ In 1996, Feder et al.⁸ identified HFE, a novel major histocompatibility complex (MHC) class I-like gene; homozygosity for a single missense mutation (C282Y) of HFE was found in 83% of the patients who were studied. Quickly, several other groups reported their findings in series of patients with hemochromatosis, and homozygosity for the C282Y mutation was found in about 85–90% of typical patients.⁹ This discovery has yielded significant benefits in clinical medicine and hepatology, including more accurate diagnosis of HFE-related HH, improved family screening, and evaluation of the role of HFE mutations in other liver diseases. Additionally, there has been a wealth of new information about the cellular and molecular mechanisms of iron homeostasis, including the discovery of the iron-regulatory hormone hepcidin.

Saint Louis University and Hereditary Hemochromatosis

I (BRB) was recruited to Saint Louis University by Coy Fitch, MD and became the Director of the Division of Gastroenterology & Hepatology in June of 1990. I went about developing the Division and recruiting faculty and fellows. I had done a quite a bit of work in the field of iron-mediated hepatotoxicity and had written several reviews and chapters on hemochromatosis with my Cleveland mentor, Tony Tavill, MD. So, when I came to St. Louis, I was known to have an interest in hemochromatosis and many patients were referred to me by hematologists, gastroenterologists, and primary care physicians to sort out iron-related problems.

In 1993, William Sly, PhD, Chairman of the Department of Biochemistry and Molecular Biology at Saint Louis University at that time came to talk to me about hemochromatosis. Dr. Sly, a brilliant biochemist and geneticist, was a member of an external scientific advisory board (SAB) for a new biotech start-up company called Mercator Genetics, based in Menlo Park, California. Supported by venture capital, Mecator Genetics had assembled a small group of exceptional biochemists, molecular geneticists, and molecular biologists. Looking for a project/challenge, Dr. Sly and others on the SAB suggested that they search for the gene for hemochromatosis.

HH is known to be a common disease (estimated to be about 1 in 250), has a long latent period before disease manifestations are apparent, has a well-established, effective treatment, and is quite well defined phenotypically. These characteristics made for an ideal candidate for possible genetic screening. Also, several academic groups from around the world (Rennes, Brisbane, LaJolla, Italy, and others) had narrowed the region of interest to the short arm of chromosome 6 in the midst of the genetically dense HLA region of the genome.

It was known that the HH gene would be near the HLA-A-3 region because of the important work of Simon and colleagues⁷ demonstrating in family studies of HH patients a strong association with HLA-A-3.

With this information in place, Dr. Sly and the other members of the SAB thought that HH would be a good project for Mecator Genetics. He asked if I would be involved as the clinician to verify the authenticity of various HH subjects. Also, I could recruit from my friends in the field as many DNA samples as possible from phenotypically well-defined HH patients.

This was certainly a wonderful opportunity which I embraced wholeheartedly. An institutional review board protocol was prepared and approved and I proceeded to obtain DNA samples from my patients and from those across the country who had a realistic and accurate diagnosis of HH.

For purposes of this study, we required that subjects have at least two of the following four parameters:

1. hepatic iron concentration >4,500 μg/g,

2. hepatic iron index (hepatic iron concentration in μmoles/g divided by age in years) of > 2.0,

3. hepatic iron staining on biopsy of 3+ or 4+, or

4. therapeutic phlebotomy of > 16 units of blood (4 g of iron).

Using these criteria, we assembled a cohort of 178 carefully characterized HH patients assembled from 32 sites across the United States with about 40 patients coming from Saint Louis University. Using DNA from this carefully selected and validated cohort of HH patients, the group at Mercator Genetics, using a positional cloning technique, identified an MHC-class-1-like gene, in which homozygosity for a single missense mutation (C282Y) was found in 83% of these subjects. This landmark work was published in Nature Genetics in August of 1996.⁸ Shortly thereafter, several research groups from around the world published their results for C282Y homozygosity in their cohorts of phenotypic HH patients and came up with very similar results.

A tremendous wealth of scientific information resulted from this finding with improved diagnosis of probands, better family studies, and several large population surveys that have clearly defined the prevalence of the C282Y homozygous state. Numerous studies have now defined the incomplete penetrance of the genetic defect demonstrating that only about 50% of C282Y homozygotes have an increased level of ferritin. (See Table 1) These studies are largely cross-sectional, but the few longitudinal studies¹⁰ suggest that for reasons, that are as yet unknown, that the majority of iron-loaded C282Y homozygotes do not progress with increased iron loading over time.

Table 1.

Population Sample	Country	n	Prevalence of homozygotes	C282Y homozygotes with a normal ferritin (%)
Electoral roll	New Zealand	1,064	1 in 213	40
Primary care	USA	1,653	1 in 276	50
Epidemiological Survey	Australia	3,011	1 in 188	25
Blood donors	Canada	4,211	1 in 327	81
General public	USA	41,038	1 in 270	33
Primary care North	America	44,082	1 in 227	25
General public	Australia	29,676	1 in 146	32
Total		124,636	1 in 240	41

The other remarkable discovery since HFE and the clinical benefits of testing for C282Y is the increased understanding of cellular and tissue regulation of iron homeostasis. A complete description of these findings is beyond the scope of this brief article but the interested reader is referred to several outstanding reviews that summarize this area in detail.^{11, 12}

Conclusion

The discovery of the gene for hemochromatosis has a soft spot at Saint Louis University. The inspirational guidance of Bill Sly was of immense value, the contact that I had with others who had carefully phenotyped HH patients was essential. The wonderful work from Mercator Genetics led to a truly major discovery. Unfortunately, as is often the case in our modern times, even though the discovery of HFE was monumental, it did not lead to a medicinal product. While the assay for “HFE mutation analysis” is frequently performed in DNA diagnostic laboratories around the world, there is not much in the way of revenue generation. So, a short time after the discovery of HFE and the Nature Genetics publication, Mercator Genetics was bought out by another biotech gene discovery company and then a year later that company was disbanded. Selling a diagnostic assay was not enough to sustain a company. Nonetheless, the scientific and clinical capital that resulted in the discovery of HFE was enormous.

Biography

Bruce R. Bacon, MD, is the James F. King MD Endowed Chair in Gastroenterology, and a Professor of Internal Medicine in the Division of Gastroenterology and Hepatology at Saint Louis University School of Medicine.

Contact: baconbr@slu.edu