Iron Overload and Liver Pathology

Vishal S. Chandan, MD

Michael S. Torbenson, MD

17.1 INTRODUCTION

The medical community has recognized that iron accumulation causes liver disease for several hundred years, but for the first 100 plus years, progress was slow in understanding the epidemiology, biology, and disease manifestations of iron overload. However, since the early 1970s, progress has been rapid, with key new insights into the genetic causes and mechanisms of iron overload as well as the normal physiology of iron metabolism. A basic understanding of normal iron metabolism helps considerably in understanding the genetic causes of iron overload, so normal iron physiology is reviewed briefly later.

Correlation with serum findings is also important. In most cases of iron overload, both the serum ferritin and the serum transferrin saturation levels are elevated. Serum ferritin levels above 1,000 µg/L and/or transferrin saturation levels of 45% or greater are widely used clinically as surrogate markers for excess body iron stores. Interestingly, there are four relatively rare conditions where these two markers do not rise in parallel, with transferrin saturation levels that remain low or normal despite significantly elevated ferritin levels: ferroportin disease, African iron overload, hereditary hyperferritinemia, and aceruloplasminemia.

17.2 OVERVIEW OF NORMAL IRON METABOLISM

A liter of healthy blood contains 0.5 g of iron, and the healthy adult body contains a total of 3 to 5 g of iron (for comparison, a USA nickel weighs 5 g). About 20 mg of iron are needed daily for ordinary physiologic functions. Most of this iron is obtained by recycling the iron found in aged and damaged red blood cells. The rest of the iron comes from dietary sources, with 1 to 2 mg needed per day. Iron is naturally present in many foods and is also a common food additive. Dietary iron can be either heme iron from meat or nonheme iron from a variety of sources, including both plants and meat.

In normal physiology, iron is important for heme synthesis, oxidative phosphorylation, DNA synthesis, and a number of other metabolic processes. Nonetheless, too much iron can generate free oxygen radicals, which are highly toxic to cells. To prevent this, the body tightly regulates iron levels in the blood and tissues. Iron levels in the body are regulated almost exclusively by controlling iron absorption in the small intestine.

Iron absorption

The major proteins and cells involved in iron metabolism are presented in Table 17.1 for quick reference. Most iron is absorbed in the duode-num and proximal jejunum. Once iron is absorbed into enterocytes, it can have several fates. If the body has sufficient iron stores, then the iron remains within the cytoplasm of the enterocytes. When the enterocytes naturally undergo apoptosis at the end of their lifespan, the iron is lost within the fecal stream, preventing iron overload. In contrast, if the body needs iron, iron is transported out of the enterocyte by ferroportin, with some help by accessory proteins including ceruloplasmin and hephaestin, and enters the blood stream, where
it is bound by transferrin and circulates within the blood. In healthy individuals, the blood contains much more transferrin protein than iron, and the transferrin proteins are approximately 30% saturated with iron. As blood iron levels increase, the excess transferrin proteins buffer the excess iron to help prevent toxicity. Saturation levels of 45% and above are associated with excess body iron stores.

Table 17.1 Important proteins and cells that play a role in iron metabolism

Protein or cells	Notes
*Proteins*
DMT-1	Dimetal transporter-1. Transports iron from gut lumen into enterocyte cytoplasm
Ferritin	Protein with an enormous capacity to bind iron and a major physiologic storage form of iron; located in the cell cytoplasm of hepatocytes, enterocytes, and Kupffer cells. Elevated serum ferritin levels are one of the most widely used surrogates for iron overload. Testing is highly sensitive (normal levels rule out iron overload) but not very specific. Ferritin levels are also used to guide phlebotomy therapy
Ferroportin	Transports iron from inside cells out to the blood (principally enterocytes and macrophages, also hepatocytes)
Hemojuvelin	Interacts with important signaling pathways (BMP, SMAD) that have hepcidin as a downstream target. Without hemojuvelin, these signaling pathways do not activate hepcidin gene synthesis in a normal fashion
Transferrin	Binds and transports iron in blood. Elevated saturation levels are (>45%) suggest increased iron stores
HFE	Mutations are the most common cause of genetic hemochromatosis. Interacts with transferrin receptor 1 and regulates hepcidin levels
*Cells*
Enterocytes	Absorption and short-term storage of iron
Hepatocytes	Major producer of ferritin and hepcidin and is the major organ for storing iron in the form of ferritin
Macrophages	Scavenges old/damage red blood cells. Another major cell type for storage of iron in the form of ferritin
*Other*
Hemosiderin	Abnormal deposits of iron

Iron movement from blood into cells

Individual cells have mechanisms to determine if they have sufficient iron stores within their cytoplasm to meet their needs. If iron is needed, cells increase expression of transferrin receptors. There are two transferrin receptors: transferrin receptor 1 is present on the membrane of all nucleated cells, whereas receptor 2 is primarily found on hepatocytes and macrophages. These membrane receptors take iron into the cells through receptor-mediated endocytosis.

Iron storage

Excess iron in hepatocytes and macrophages is incorporated into ferritin molecules for storage. Ferritin can hold up to 4,500 atoms of iron per ferritin protein complex. Serum ferritin levels above 1,000 µg/L are widely used as a surrogate marker for excess body iron stores. Ferritin in the liver is not visible on Perls stain in most cases, but occasionally ferritin is seen as a faint blue blush in the hepatocyte cytoplasm. The iron in ferritin can be rapidly accessed to meet physiologic needs. If body iron levels are excessive over a sufficiently long period of time, hemosiderin deposits develop.

Hemosiderin deposits are granular and golden brown on hematoxylin and eosin (H&E) stains. They are made of iron, degraded ferritin, and small amounts of various proteins. In contrast to ferritin, the iron in hemosiderin is not readily available for biologic needs. Hemosiderin in both genetic and nongenetic iron overload is identical on H&E and Perls iron stain, but they differ significantly in both the metal composition and the organic components.¹

The central role of hepcidin in regulation of iron levels

When blood iron levels are low, iron is released from the ferritin stored in enterocytes, hepatocytes, and macrophages into the blood. When blood levels are adequate or high, hepcidin prevents these cells from releasing iron into the blood. Hepcidin accomplishes this in part by degrading ferroportin,² which is the key protein involved in transporting iron out of cells into the blood. In contrast, hepcidin levels decline in response to low iron levels, allowing increased release of iron into the blood from enteric cells, hepatocytes, and macrophages. Hepcidin (encoded by HAMP) is an acute phase reactant produced by hepatocytes³ and biliary epithelium.⁴ Because it is an acute phase reactant, hepcidin levels can be elevated in a variety of inflammatory and infectious conditions, leading to anemia of chronic disease.

Hepcidin plays a central role in all known forms of genetic hemochromatosis, with each mutation leading to impaired hepcidin production or function.⁵,⁶ The loss of production or function of hepcidin in turn gradually leads to excess iron absorption, with subsequent iron deposition in the liver and other organs. The major causes of genetic hemochromatosis are discussed individually below and are summarized in Table 17.2.

In contrast to hemochromatosis that results from loss of hepcidin function, rare mutations have also been described that increase hepcidin function, with clinical manifestations of congenital refractory anemia.⁷ Increased expression of hepcidin (with subsequent development of anemia) has also been reported in a hepatic adenoma.⁸ Similarly, hepatic adenomas occurring in individuals with Type 1a glycogen storage disease are often associated with anemia that resolves after the adenoma is resected,⁹ implying hepcidin is overexpressed by the adenomas. In contrast, hepatocellular carcinomas tend to have lower levels of hepcidin expression.¹⁰

17.3 HEMOCHROMATOSIS

History of hemochromatosis as a disease

Armand Trousseau (1801 to 1867), a French internist, reported the syndrome of liver cirrhosis, pancreas fibrosis, and cutaneous hyperpigmentation in 1865,¹¹ shortly before his own death from pancreas carcinoma. However, it appears that he did not recognize the role of iron in the disease process. Another French physician, Troisier, described a case of “diabète bronzé et cirrhose pigmentaire” in 1871 and both confirmed the observation of Trousseau and demonstrated iron in various tissues.¹² In fact, Troisier’s triad of diabetes, skin hyperpigmentation, and cirrhosis became the working definition for hemochromatosis for many decades. Another milestone occurred in 1935 when Sheldon, an English gerontologist, reviewed the literature of 300+ cases of hemochromatosis and concluded that iron accumulation was likely the result of increased absorption.¹³ Other physicians and scientists before and after Sheldon’s seminal paper thought the iron accumulation in hemochromatosis might be secondary to hemorrhage or other sources of tissue injury. Sheldon’s paper also suggested hemochromatosis had a genetic
basis. A genetic link was subsequently confirmed in 1975 by Simon and colleagues in a study that demonstrated a link between the clinical hemochromatosis phenotype and a human leukocyte antigen-A locus on chromosome 6p, with an inheritance pattern most consistent with an autosomal recessive disease.¹⁴ The specific gene causing most forms of hemochromatosis was discovered in 1996¹⁵ and eventually named HFE, with the H shorthand for “high” and “FE” for iron. Subsequent studies identified cases of iron overload that were not caused by HFE mutations, leading to a gradual recognition of additional genes important in iron metabolism. The discovery of Hepcidin (coded by HAMP) by several groups in 2000¹⁶ and 2001³,¹⁷ eventually led to a unifying mechanism for iron genetic iron overload disease: impairment of hepcidin function.

Table 17.2 Summary of genetic iron diseases involving the liver

Disease	Gene (protein)	Inheritance	Primary ethnicity	Onset	Principal iron location
Hemochromatosis Type 1	HFE	Recessive	Northern European	Late	Hepatocytes > Kupffer cells
Juvenile hemochromatosis Type 2A	HFE2 (also known as HJV)	Recessive	European	Early	Hepatocytes > Kupffer cells
Juvenile hemochromatosis Type 2B	HAMP (hepcidin)	Recessive	European	Early	Hepatocytes > Kupffer cells
TFR2-associated hemochromatosis	TFR2	Recessive	European Asian	Late	Hepatocytes > Kupffer cells
Hemochromatosis	SCL11A2 (DMT-1)	Recessive	European	Early	Hepatocytes > Kupffer cells
Ferroportin disease	SLC40A1 (ferroportin, loss of function mutation)	Dominant	Pan-ethnic	Late	Kupffer cells > hepatocytes
Ferroportin associated hemochromatosis (previously referred to as ferroportin disease type B)	SLC40A1 (ferroportin, activating mutation)	Dominant	Pan-ethnic	Late	Hepatocytes > Kupffer cells
Hypotransferrinemia	TF (Transferrin)	Recessive	European Asian	Early	Kupffer cells > hepatocytes
Hypoceruloplasminemia	CP (Ceruloplasmin)	Recessive	European Asian	Late

17.4 HFE MUTATIONS

Clinical and epidemiologic findings

More than 37 HFE mutations have been reported,¹⁸ but C282Y and H63D mutations are by far the most numerically and clinically important. C282Y mutations are strongly linked to northern European genetic ancestry,¹⁸ whereas H63D mutations have a wider ethnic distribution.¹⁹ Approximately 35% of individuals with northern European ancestry will have an HFE mutation (Table 17.3). In contrast, other ethnic groups have much lower frequencies of HFE mutations (Table 17.4).

The normal HFE protein is expressed principally in cells that traffic iron, such as duodenal crypt cells,
hepatocytes, and Kupffer cells. Mutations in the HFE gene make the HFE protein unstable by disrupting disulfide bonds that are important for HFE binding to b2-microglobulin. The mutations also impair a key signaling pathway in hepcidin synthesis, known as the bone morphogenic protein signaling pathway.

Table 17.3 HFE mutations in northern European populations

Genetic status	Population (percent affected)
C282Y heterozygote	9
C282Y homozygote	0.5
H63D heterozygote	22
H63D homozygote	2.0
C282Y/H63D compound heterozygote	2.0
Wild/wild	65
From Hanson, E.H., G. Imperatore, and W. Burke, HFE gene and hereditary hemochromatosis: a HuGE review. Human Genome Epidemiology. Am J Epidemiol, 2001. 154(3): p. 193-206, by permission of Johns Hopkins Bloomberg School of Public Health.

Table 17.4 HFE mutations in other populations

Genetic status	Hispanic (% affected)	Black (% affected)	Pacific islander (% affected)	Asian (% affected)
C282Y heterozygote	3	2	2	0.1
C282Y homozygote	0.06	0.01	0.00	0.00
C282Y/H63D compound heterozygote	0.39	0.13	0.00	0.00
Data from Crownover, B.K. and C.J. Covey, Hereditary hemochromatosis. Am Fam Physician, 2013. 87(3): p. 183-90.

Homozygous C282Y mutations in the HFE gene account for 81% of clinical genetic hemochromatosis cases, whereas compound C282Y and H63D mutations are seen in 5% of cases.²⁰ Homozygosity for H63D mutations appears to lead to iron overload principally in the setting of other chronic liver diseases. Other mutations, such as S65C, have also been linked to iron accumulation²¹,²² but their role in iron overloading is less clear.²³ S65C mutations appear to be enriched in populations from Britanny, France.²⁰ It seems likely that H63D, S65C, and other mutations play a synergistic role for excess iron accumulation in the setting of other disease processes or genetic polymorphisms, but by themselves are insufficient drivers of iron overload and do not directly cause hemochromatosis. They can contribute to iron overload disease when inherited along with a C282Y mutation. Individuals with C282Y mutations are at much higher risk for iron accumulation than individuals with H63D mutations and C282Y homozygotes have greater risk for iron accumulation than C282Y heterozygotes. An important point, however, is that there is great phenotypic variation, even for individuals who are homozygous for C282Y mutations. In fact, a meta-analysis found that the overall clinical disease penetrance of C282Y homozygosity was only 13.5%.²⁰ Disease penetrance was higher when defined as having at least mild iron on liver biopsy, at 42% for C282Y homozygous men and 19% for homozygous women.²⁰ The tremendous variation in disease penetrance appears to result from gender, environmental factors, dietary factors, and genetic polymorphisms.²⁴

Morbidity and mortality with HFE-related hemochromatosis

Individuals with HFE hemochromatosis are at increased risk for liver cirrhosis and for hepatocellular carcinoma.²⁵, ²⁶, ²⁷, ²⁸ Heart failure and diabetes also contribute to morbidity and mortality because of iron deposition in the heart and the pancreatic islet cells. Cardiovascular disease continues to be an important cause of morbidity and mortality even after liver transplantation.²⁹ Also of note, a significant proportion of the diabetes risk may result from genetic changes that cosegregate with HFE mutations, but are not directly related to iron overload in the islet cells.³⁰ Joint disease can affect both heterozygote and homozygote individuals with HFE mutations. The second and third metacarpal-phalange joints and the interphalangeal joints can develop arthropathy because of iron deposits in the cartilage and synovial cells. Infections with Vibrio vulnificus, a virulent bacterium that is highly dependent on iron, are also increased in individuals with iron overload.³⁰ Infection can be caused by eating undercooked seafood, especially from the Gulf of Mexico.

Clinical indications for liver biopsy in individuals with HFE mutations

The European Association for the Study of the Liver (EASL) guidelines recommend liver biopsies in C282Y homozygous individuals to assess the degree of fibrosis when serum ferritin levels are above 1,000 µg/L, or there are elevated aspartate aminotransferase (AST) levels, hepatomegaly, or age over 40 years. The American Association for the Study of Liver Diseases (AASLD) guidelines are broadly similar and recommend biopsies to stage fibrosis for C282Y homozygotes or compound heterozygotes when ferritin is >1,000 µg/L or there are elevated serum levels of AST or alanine aminotransferase (ALT).

Impact of HFE mutations on other chronic diseases

As noted previously, the disease penetrance of HFE mutations is influenced by complex genetic, environmental, and dietary variables.²⁴ In this regard, several other chronic liver diseases can affect HFE penetrance,
including chronic viral hepatitis C and B, alcohol-related liver disease, nonalcoholic fatty liver disease (NAFLD), and α-1-antitrypsin deficiency. Although the data are incomplete and substantially mixed, the general trend suggests that HFE mutations predispose to iron accumulation in other chronic liver diseases and become increasingly penetrant as fibrosis progresses. As one example, cirrhotic livers with both α-1-antitrypsin deficiency and marked iron overload are enriched for HFE mutations.²¹ There are many negative studies that found no association between iron accumulation and fibrosis stage in other chronic liver diseases, most likely reflecting the difficulty of identifying a modest effect in the very complex setting of clinical cohort studies, where it is very difficult to adequately control for all of the factors that have been reported to influence both iron status as well as fibrosis risk.

17.5 JUVENILE HEMOCHROMATOSIS (USUALLY CHILDREN/EARLY ONSET)

Juvenile hemochromatosis is a rare cause of hemochromatosis. There are two subtypes. Subtype 2A results from mutations in HFE2, which encodes the protein hemojuvelin. Subtype 2B results from mutations in HAMP, which codes for hepcidin. Overall, Type 2A is more common than Type 2B.³¹,³² Hemojuvelin disease typically presents with impotence or amenorrhea and not with liver or joint disease. Cardiomyopathy is also common at presentation. In the liver, there can be marked hepatocellular iron overload. The disease tends to be more aggressive than HFE-related hemochromatosis, typically running a rapidly progressive clinical course.³³ G320V mutations are the most common mutation in juvenile hemochromatosis, found in 80% to 90% of cases, but about 30 total mutations in HFE2 have been reported to date. Juvenile hemochromatosis caused by HAMP mutations also has marked hepatocellular iron overload and typically runs a severe clinical course. Approximately 12 different mutations have been reported to date. Hypogonadism and cardiac disease are prominent clinical manifestations.

17.6 TRANSFERRIN RECEPTOR GENE 2 (USUALLY ADULTS/LATE ONSET)

This rare form of genetic iron overload was historically called type 3 hemochromatosis and was initially described in Italian patients with systemic iron overload who were negative for HFE mutations.³⁴ Subsequent studies identified mutations in the TFR2 gene,³⁵ with about 20 mutations identified to date. The disease has a variable clinical course, but there can be marked hepatocellular iron deposition and systemic iron overload. Also of note, in the general asymptomatic adult population, polymorphisms in TRF2 are common and can lead to mild increases in blood iron levels without overt iron overload disease.³⁶

17.7 DMT-1 MUTATIONS (USUALLY OLDER CHILDREN)

This very rare disease is caused by mutations in the SCL11A2 gene. Few cases have been reported, and data are quite limited.³⁷,³⁸ Children present with severe microcytic anemia. Iron accumulation is primarily in hepatocytes and can be marked, but biopsies may be negative for iron in very young children.

17.8 FERRITIN MUTATIONS

Ferritin has both light and heavy chains, encoded by FTL and FTH genes. Very rare mutations in both genes have been reported. In general, mutations affecting the ferritin genes are associated with high serum ferritin levels but low transferrin saturation levels.

Mutations in the ferritin light chain gene, FTL, lead to both the hereditary hyperferritinemia cataract syndrome and to hereditary neuroferritinopathy. Individuals affected by the hereditary hyperferritinemia cataract syndrome have elevated serum ferritin levels with early onset cataract formation. Individuals with hereditary neuroferritinopathy have iron accumulation in the central nervous system, particularly the basal ganglia, which leads to neurodegenerative dysfunction. There is very little published data on liver histology, but there appears to be no iron accumulation in either of the diseases caused by FTL mutations, though the hepatocytes in hereditary neuroferritinopathy can have pale nuclear inclusions on H&E that stain light blue on Perls iron stain.³⁹,⁴⁰ Mutations in the heavy gene, FTH1, do lead to iron accumulation and there can be moderate iron overload with a Zone 1 predominant pattern.⁴¹ Iron can also accumulate in the splenic macrophages.⁴¹

17.9 TRANSFERRIN MUTATIONS

Mutations in the transferrin gene, TF, are very rare. A complete or nearly complete absence of transferrin protein leads to clinically significant disease in childhood.⁴², ⁴³, ⁴⁴ The disease appears to be autosomal recessively inherited.⁴⁵ Individuals have very low or absent transferrin levels, severe hypochromic microcytic anemia, and marked hepatic iron overload.⁴², ⁴³, ⁴⁴, ⁴⁵ Histologic
descriptions are scant, but marked iron deposition is seen in both hepatocytes and Kupffer cells.⁴⁶,⁴⁷

In contrast, heterozygosity for TF mutations can lead to haploinsufficiency in terms of red blood cell production, but only leads to liver iron overload if other genetic mutations, such as HFE, are also present.⁴⁸

17.10 CERULOPLASMIN MUTATIONS (CHILDREN AND ADULTS)

Ceruloplasmin, encoded by the CP gene, is the major protein for transporting copper in the blood. However, it also plays a role in iron metabolism, and inactivating mutations can lead to marked iron overload in the liver, pancreatic islet cells (leading to diabetes), and brain (leading to ataxia and dementia).⁴⁹,⁵⁰ Aceruloplasminemia shows a wide age range at presentation, from late teenage years to elderly. Overall, most cases are diagnosed in later years, when individuals present with neurologic symptoms. However, younger individuals can present with anemia, mild liver enzyme elevations, or with blood iron test abnormalities.⁵¹,⁵² In general, blood testing shows elevated serum ferritin levels, but low serum iron levels and low transferrin saturation. In the liver, iron accumulation is found predominately in hepatocytes and to a lesser degree in Kupffer cells. In the lobules, the hepatic iron deposition tends to have a panlobular distribution and pericanalicular pattern.⁵³, ⁵⁴, ⁵⁵, ⁵⁶, ⁵⁷

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