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Update and Application to Liver Biopsy Interpretation in Clinical Practice
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Case 4 -
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Hereditary Hemochromatosis
Julia C. Iezzoni
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Clinical History
The patient, a 28-year-old Caucasian male, was noted to
have mildly elevated aminotransferases during a routine examination performed for insurance purposes.
While viral studies and autoantibodies were negative, his serum iron level was elevated. A fasting
transferrin saturation and serum ferritin level were obtained, and both were increased. Genetic testing
demonstrated C282Y homozygosity for the HFE gene. A liver biopsy was
performed.
Diagnosis: Hereditary Hemochromatosis
Pathologic findings: The liver biopsy demonstrates gold-brown
colored granules in the cytoplasm of the hepatocytes. With the Perl's Prussian blue stain, these
granules are identified as iron. The iron demonstrates a marked zonal pattern of distribution, with
involvement of the periportal hepatocytes and sparing of zones 2 and 3.
Discussion
Hereditary hemochromatosis (HH), an autosomal recessive genetic disorder of iron
metabolism, is characterized by malfunction of the regulation of iron absorption by the intestine that
results in chronic excessive uptake of dietary iron. The excess iron deposits in the parenchymal cells
of the liver, heart, pancreas, joints, pituitary gland, and other tissues, causing cell and organ
damage. If left untreated, this progressive injury eventually results in multiorgan dysfunction or
failure. Hereditary hemochromatosis is common; specifically, it is the most commonly identified genetic
disease in Caucasians. It is particularly prevalent in individuals of northern European ancestry, in
whom it found in 1 in 250 individuals, and the frequency of heterozygosity in this population is
approximately 1 in 10 individuals. The recent identification and cloning of
HFE, the gene associated with most cases of HH, has improved the understanding of both normal and
abnormal iron metabolism. In addition, the development of a commercial, widely available, genetic test
for the HFE gene has modified the indications for liver biopsy in the
diagnosis and management of patients with abnormal serum iron studies. As such, these recent advances in
understanding the pathogenesis of HH and the role of the liver biopsy in the evaluation of patients with
iron overload will be discussed.
HFE, the recently identified gene associated with HH, is
located on the short arm of chromosome 6 (6p). The most common genetic defect that causes HH is a
missense mutation of the HFE gene that leads to the substitution of tyrosine
for cysteine at amino acid position 282 (C282Y). While the prevalence of this mutation varies with the
ethnicity of the population being studied, overall, C282Y/C282Y homozygosity has been found in
approximately 90% of individuals with phenotypic HH. The second most common genetic abnormality of the
HFE gene that results in HH is a missense mutation in which aspartic acid
is substituted for histidine at amino acid position 63 (H63D). Although this mutation appears to have
little effect when inherited alone, it contributes to disease expression when inherited along with the
C282Y mutation. This C282Y/H63D compound heterozygosity accounts for 3% to 5% of cases of HH. The
remaining cases of phenotypical HH are due to either rare additional mutations of the HFE gene or abnormalities of other genes that encode proteins necessary for normal
iron metabolism (e.g. transferrin receptor, ß2-microglobulin).
While it is known that iron balance in the body is regulated primarily at the level of
the intestinal epithelium through its control of absorption of dietary iron, many of the details of iron
homeostasis remain unclarified. The recent identification of the HFE gene,
however, has helped to elucidate the biology of iron metabolism and its altered regulation in HH. The
protein product of the HFE gene is a 343-residue transmembrane glycoprotein
that resembles the MHC Class I proteins in its sequence and three-dimensional structure. This protein
has a large extracellular domain, a single transmembrane region, and a short cytoplasmic tail. The HFE
protein requires interaction, through noncovalent binding, with ß2-microglobulin for normal presentation
on the surface of cells. Once at the cell surface, the HFE protein is thought to facilitate iron uptake
from the bloodstream by a mechanism that involves pH-dependent binding to transferrin receptor (TfR), the
membrane receptor for the serum iron carrier protein, transferrin (Tf). According to this model, cells
acquire iron from the bloodstream in the form of diferric transferrin (Tf-Fe). At the cell surface pH of
7.4, TfR binds iron-rich Tf-Fe. The TfR:Tf-Fe complex then enters the cell by receptor-mediated
endocytosis. At the acidic pH (6.2) of the endosomes, the iron is released from Tf and is transported to
the cytosol, where it is used to meet the cell's metabolic needs or is incorporated into the iron-storage
protein, ferritin. The Tf and TfR proteins then cycle back to the cell surface. In HH, it is thought
that the mutant form of the HFE protein may lose the ability to facilitate TfR-dependent serum iron
uptake into the intestinal epithelial cells, leading to a relative iron deficiency in these cells. This
may result in increase expression of the iron transport protein, divalent metal ion transporter 1
(DMT-1), which is responsible for dietary iron absorption by the small intestinal epithelial cells. In
turn, this upregulation of DMT-1 results in increased absorption of dietary iron.
With the discovery of the HFE gene, it is tempting to
propose that a diagnosis of HH be based on genotype. This approach, however, would not account for the
highly variable clinical expression (i.e. phenotype) of the disease. Among affected individuals, the
clinical presentation ranges from mild, if any, symptoms to life-threatening heart and liver disease.
This inconstant phenotype is due in part to the variable penetrance and allelic heterogeneity of the
HFE mutations. While C282Y homozygosity appears to have a high predictive
accuracy for the HH phenotype, as defined by the finding of elevated transferrin saturation, in contrast,
full disease expression, characterized by progressive tissue iron overload, occurs in only 58% of these
homozygotes. Furthermore, the H63D mutation appears to cause liver disease only when it is present as a
compound heterozygote (C282Y/H63D), and the compound heterozygote's risk for the development of symptoms
is less than that for C282Y homozygosity. As such, the penetrance and disease course of the mutant forms
of the HFE gene are variable. The HH phenotype also is affected by
physiologic and pathologic factors that influence iron stores. Of these, gender is most significant.
Disease expression is markedly decreased in women, presumably due to the protective effect of iron loss
during menstruation and pregnancy. Age also has an impact on phenotype, due to the progressive
accumulation of iron and resultant tissue damage that occurs over time. Increased dietary iron, vitamin
C, which is an enhancer of iron uptake, and chronic alcohol abuse may increase the amount of iron
overload, and thereby increase the tissue damage and subsequent likelihood of symptoms. Conversely,
conditions that result in iron loss, such as chronic gastrointestinal bleeding due to peptic ulcer
disease or regular blood donation, also are protective against the development of HH symptoms. As such,
HH genotype and phenotype are not congruent. Current models of HH propose that the HFE gene mutants confer a risk for disease development and interaction with
modifying factors contributes to the phenotypic expression of the disease.
Despite the difficulties in defining what constitutes a diagnosis of HH, it is important
for the diagnosis not to be missed as it is a readily treatable disease, especially when detected early
in its course. For a variety of reasons, however, HH is still unrecognized and under-diagnosed. Early
in the disease course, patients are either asymptomatic or manifest symptoms that are mild and
non-specific (e.g. lethargy, fatigue). Later in the disease course, when patients present with symptoms
referable to tissue and organ damage (e.g. cirrhosis, diabetes, arthritis, congestive heart failure), the
diagnosis may not be considered as many of the symptoms and signs are indicative of disease processes
other than HH. Furthermore, despite the abundant data that establishes HH as a common genetic
abnormality, delayed diagnosis may come as a result of a physician not considering HH as a possibility
under the misperception that it is a rare disorder.
Once the diagnosis of HH is considered, however, the evaluation is relatively
straightfoward. To this end, diagnostic algorithms have been proposed to evaluate patients for possible
HH (Figure 1). This evaluation frequently incorporates HFE gene mutation
analysis, and with the availability of this genetic test, a liver biopsy is not always considered
necessary to establish the diagnosis of HH. As will be discussed below, in certain clinical
circumstances, the liver biopsy is an important part of the evaluation and management patients with iron
overload.
The C282Y mutation also has been detected in patients with a variety of chronic liver
diseases other than HH. In these patients, it is hypothesized that the increased iron stores act
synergistically to enhance disease development or progression. For example, 40% to 50% of patients with
porphyria cutanea tarda are reported to have at least one allele with the C282Y mutation, and many
patients are C282Y homozygotes. Some studies of non-alcoholic steatohepatitis (NASH) have demonstrated
that approximately 40% of these patients have at least one allele with the C282Y mutation. Furthermore,
the NASH patients who are C282Y positive may have a higher frequency of fibrosis, indicating a possible
synergistic effect between tissue iron stores and the development of fibrotic liver disease in patients
with NASH. Similarly, some investigations of patients with chronic hepatitis C have reported that the
presence of HFE gene mutations (especially C282Y) is associated with
increased fibrosis, but other studies have not found this association. While additional investigations
are necessary to determine the role (if any) of iron overload in other hepatic disorders, these findings
raise the question of whether genetic testing for abnormalities of the HFE
gene should be performed routinely on patients with other forms of chronic liver disease.
Figure 1: Proposed algorithm for the evaluation for possible
hereditary hemochromatosis (From Bacon BR 2001)
Pathology
While the availability of genetic testing for HFE gene
mutations has decreased the indications for liver biopsy in the evaluation of patients with possible HH,
in certain clinical circumstances, histologic examination of liver tissue is an important part of the
evaluation and management of patients with iron overload. In particular, in patients who are non-C282Y
homozygotes but have a phenotype compatible with hemochromatosis, a liver biopsy is indicated to evaluate
for other causes of iron overload. As described below, certain histologic patterns of hepatic iron
deposition provide clues as to the etiology of the iron overload. In addition, a liver biopsy is
necessary to assess the degree of fibrosis and determine whether or not cirrhosis is present. From a
prognostic standpoint, the presence of fibrosis or cirrhosis is important to establish, as the risk of
hepatocellular carcinoma is significantly increased in those patients who have established cirrhosis. In
this setting, the identification of so-called iron-free-foci (small discrete regions devoid of
hepatocellular iron) is of importance, as these may be an early step in the development of hepatocellular
carcinoma.
Of note, the role of the hepatic iron index (HII), a quantitative determination of
hepatic iron content, is less important in the diagnosis of HH due to the availability of genetic testing
for the HFE gene. Numerous previous studies showed that HII >1.9 is
consistent with homozygous HH. However, recent studies have shown that as many as 15% of patients with
HH (identified as C282Y homozygosity) will have an HII of <1.9.
Microscopic evaluation of the liver biopsy in patients with possible iron overload
includes a semi-quantitative assessment of the amount of iron deposition and a qualitative assessment of
the pattern of iron distribution in the different cell types and zones within the hepatic lobules.
Typically, Perl's Prussian blue is the histochemical stain that is used to demonstrate iron, as it
identifies both storage forms of intracellular iron, ferritin and hemosiderin.
A variety of different schemes have been proposed for the semi-quantitative analysis of
the amount of hepatic iron observed microscopically. One of the most widely used is that of Searle et al
(Table 1). With this system, the amount of hepatic iron is assigned a grade based on the ease in which
it can be discerned microscopically, and there is no discrimination made based on the cell type in which
the iron is observed.
Table 1: Histologic grading of iron stores
| Grade | Ease of observation; magnification (eyepiece x objective) required for identification |
| 0 | Granules absent or barely discernible; X400 |
| 1+ | Barely discernable; X250
Easily confirmed; X400 |
| 2+ | Discrete granules resolved; X100 |
| 3+ | Discrete granules resolved; X25 |
| 4+ | Masses visible; X10 or naked eye |
Qualitative assessment of the pattern of iron distribution in the different cell types
and zones within the hepatic lobules provides clues as to the etiology of the iron overload. These
patterns have been identified as the parenchymal pattern, the mesenchymal pattern, and the mixed
pattern.
The parenchymal pattern corresponds to that of HH. Iron is found predominantly within
hepatocytes, in a distinct pericanlicular location in the cells. Early in the disease, this iron is seen
only in zone 1 hepatocytes. As iron continues to accumulate, all of the hepatocytes acquire iron, with a
decreasing periportal-to-centrilobular gradient. Eventually, iron pigment also accumulates in bile duct
epithelium, Kupffer cells, portal macrophages, and vascular endothelium, thereby assuming a mixed pattern
of distribution (see below).
In the mesenchymal pattern, iron deposition occurs predominately in sinusoidal cells
(e.g. Kupffer cells, sinusoidal endothelial cells, and stellate cells). When present, parenchymal iron
deposition is scarce and mainly located in hepatocytes that are adjacent to clusters of iron-laden
macrophages. The mesenchymal pattern is seen in iron overload due to blood transfusions, dietary iron
excess, and long-term hemodialysis.
Mixed pattern corresponds to more complex conditions, as encountered in patients with
several sources of iron overload (e.g. hemochromatosis and chronic alcohol abuse).
Of note, in a recent study that evaluated the usefulness of the histological pattern of
iron deposition in determining the probability of an iron-loaded patient having HFE-related iron
overload. This study demonstrated that a more mesenchymal-type pattern reliably predicts the absence of
homozygosity for the C282Y mutation. However, a HH-type pattern of iron deposition may be seen in other
forms of liver disease besides HH and is not predictive of either C282Y homozygosity or compound
heterozygosity.
References
- Bacon BR. Hemochromatosis: Diagnosis and management. Gastroenterol 2001;120:718-25.
- Brunt EM, Olynyk JK, Britton RS, Janney CG, Di Bisceglie AM, Bacon BR. Histological evaluation of iron in liver biopsies: Relationship to HFE mutations. Am J Gastroenterol 2000;95:1788-93.
- Lyon E, Frank EL. Hereditary hemochromatosis since discovery of the HFE gene. Clin Chem 2001;47:1147-56.
- Searle J, Kerr JFR, Halliday JW, Powell LW. Iron storage disease. In: Macsweem RNM, Anthony PP, Scheurer PJ, Burt AD, Portmann BC, eds. Pathology of the Liver. London: Churchill Livingstone, 1994:219-41.
- Tavill AS. Diagnosis and management of hemochromatosis. Hepatol 2001;33:1321-8.
- Turlin B, Deugnier Y. Evaluation and interpretation of iron in the liver. Sem Diag Path 1998;15:237-45.
- Searle J, Kerr JFR, Halliday JW, Powell LW. Iron storage disease. In: Macsweem RNM, Anthony PP, Scheurer PJ, Burt AD, Portmann BC, eds. Pathology of the Liver. London: Churchill Livingstone, 1994:219-41.
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