Hemochromatosis and HFE mutations
The “classical” type of familial hemochromatosis that is transmitted as an autosomal recessive disorder is usually due to homozygosity for the C282Y mutation of the HFE gene. It is expected that mutations of HFE cause the great majority of the cases of heritable iron overload in humans. There are at least 37 known mutations of the HFE gene (Table 8.1) (Chapter 4). Some individuals who are homozygous for any of the known mutations may develop heavy iron overload. At least six mutations are associated with mild iron accumulation; and at least six mutations were discovered in patients who did not have iron overload. There is insufficient reported information in the literature to determine if six of the mutations are sufficiently deleterious to result in iron overload. Approximately 1500 mutations of the cystic fibrosis gene (CF) are known, and the gene encodes a 1480 amino acid protein. In contrast, HFE is much smaller than CF, and encodes a protein of only 343 amino acids. Thus, it is expected that fewer mutations of HFE than mutations of CF will be eventually discovered.
Although the autosomal recessive inheritance pattern of “classical” hemochromatosis had long been recognized, identification of the responsible gene remained elusive for many years (Table 8.1). An important breakthrough in the search for the gene occurred in 1976 when hemochromatosis was found to be linked closely to the human leukocyte antigen (HLA)-A*03 region of the short arm of chromosome 6.
Localized iron overload can sometimes occur in the lungs (pulmonary hemosiderosis) and the kidneys (renal hemosiderosis).
Idiopathic pulmonary hemosiderosis (IPH) (also known as Ceelen–Gellerstedt syndrome) is a rare disorder of unknown etiology characterized by recurrent episodes of diffuse alveolar hemorrhage and accumulation of storage iron in the lung parenchyma. It is most common in children (age 1 to 7 years) but can also occur in adults. Clinical manifestations of IPH include pulmonary symptoms (hemoptysis, dyspnea, cough), parenchymal lesions on chest X-ray, and iron deficiency anemia of unknown cause. Diagnosis depends on exclusion of other disorders, such as inflammatory pulmonary capillaritis, in which diffuse alveolar hemorrhage is a cardinal sign. The clinical course of IPH is variable; in chronic cases, the localized iron overload can result in pulmonary fibrosis, and death can sometimes occur due to pulmonary hemorrhage. Treatment includes supportive therapy and administration of corticosteroids that can be combined with other immunosuppressive agents, such as azathioprine. Successful resolution of some cases of IPH with immunosuppressive drugs suggests that an immunologic mechanism could be involved in the pulmonary capillary damage underlying alveolar bleeding which, in turn, leads to pulmonary iron accumulation.
Hematite miners and other workers chronically exposed to iron ore dust may develop iron overload of the lungs and adjacent lymph nodes, but serum iron measures are usually normal.
Marked iron accumulation in the kidneys is rare in hemochromatosis, but when present, storage iron is usually located in cells of the tubules, particularly the convoluted tubules.
The C282Y polymorphism of the HFE gene on chromosome 6p21.3 is the most common known human mutation that has a marked effect on iron absorption and homeostasis. Approximately 12% of Caucasians of northern or western European descent living in Europe or in derivative countries such as the US, Canada, Australia, or New Zealand are simple heterozygotes for C282Y, and they outnumber C282Y homozygotes in these populations by approximately 25:1. In the US alone, approximately 20 million whites are C282Y heterozygotes. If iron-related organ injury or another deleterious allele on C282Y-bearing chromosome 6p haplotypes were to occur in C282Y heterozygotes, the number of individuals at potential risk is great. This has led to an interest in defining: (a) the iron phenotype and any related iron or liver morbidity of C282Y heterozygotes and their management; (b) the optimal means to identify C282Y heterozygotes and to estimate their prevalence in populations; and (c) the association of C282Y with various non-iron-related disorders.
The HFE C282Y mutation arose in northwestern Europe, perhaps in the Neolithic Age. The original C282Y mutation probably occurred on a chromosome 6 haplotype characterized by human leukocyte antigens (HLA)-A*03, B*07, and by the marker allele D6S105(8). C282Y spread with various population movements, especially Viking migrations. Many C282Y homozygotes alive today have one or two copies of the ancestral haplotype. Likewise, all C282Y heterozygotes have inherited a common HFE polymorphism, but also much or all of an ancestral chromosome with its other component genes and alleles, the effects of which must be considered in understanding fully the effects of C282Y heterozygosity.
TFR2 hemochromatosis (OMIM #604250) is a rare autosomal recessive disorder characterized by elevated serum iron measures, parenchymal iron deposition, and complications of iron overload. In some kinships, severe iron overload occurs in children or young adults. In individual cases, the TFR2 hemochromatosis phenotype may resemble that of HFE hemochromatosis or HJVhemochromatosis (Chapter 8).
In 1999, Kawabata and colleagues cloned and sequenced a human gene homologous to the TFR gene that encodes classical transferrin receptor (TFR1). They named the newly discovered gene TFR2 (OMIM *604720), and mapped it to chromosome 7q22. Two transcripts (alpha and beta) are expressed from this gene; the alpha transcript is expressed predominantly in the liver. TFR2-alpha is a second transferrin receptor that mediates cellular iron transport in vitro. In normal subjects, most iron uptake by the liver is transferrin mediated. Expression of TFR1 in hepatocytes, as in other non-reticuloendothelial cell types, is down-regulated in response to increased intracellular iron. Consequently, hepatocyte TFR1 is undetectable in patients with HFE hemochromatosis and hepatic iron loading. Nonetheless, hepatic iron loading in HFE hemochromatosis is progressive. Experiments in mice demonstrate that TFR2 makes only a minor contribution to the uptake of transferrin-bound iron by the liver, but rather TFR2 is thought to modulate the signaling pathway that controls hepcidin expression. In 2000, Camaschella and colleagues described persons with hemochromatosis phenotypes in two unrelated Sicilian families who had mutations in TFR2.
The age of onset and severity of iron overload varies moderately in patients with TFR2 hemochromatosis.
Hereditary atransferrinemia (OMIM #209300) is a rare disorder characterized by severe quantitative or functional deficiency of transferrin. As a consequence, there is reduced delivery of iron to erythroid cells in the marrow, reduced hemoglobin synthesis, increased iron absorption, and severe iron overload of parenchymal organs.
In 1961, Heilmeyer and colleagues described atransferrinemia in a girl who had severe hypochromic anemia at age 3 months and severe, progressive generalized iron overload. Patients from other countries with similar abnormalities have been reported subsequently, and explanatory mutations in the gene that encodes transferrin (TF; chromosome 3q21) have been demonstrated in four cases. A similar disorder discovered in inbred mice is due to a splice-site mutation in Tf, the ortholog of TF in humans. Cases of acquired or secondary atransferrinemia or hypotransferrinemia have also been described in patients with diverse underlying conditions.
Manifestations of anemia are the most common clinical abnormalities in patients with hereditary atransferrinemia (Table 19.1). Several probands have had pallor, fatigue, or severe hypochromic, microcytic anemia at birth or in infancy. An Italian infant also had hypovolemia, metabolic acidosis, and persistent fetal circulation. Pallor and anemia were discovered for the first time at age 7 years in a patient from Japan, and at age 20 years in a patient from the US. Most patients have had a systolic ejection murmur attributed to chronic anemia. One patient had mild hepatomegaly.
Divalent metal transporter-1 (DMT1) is a member of the “natural-resistance-associated macrophage protein” (Nramp) family. DMT1 is upregulated by dietary iron deficiency, is expressed strongly on the microvillus membranes of duodenal enterocytes at the villus tips, and is a key mediator of iron absorption. DMT1 also mediates iron transfer from endosomes into the cytosol of developing erythroid cells. The SLC11A2 gene that encodes DMT1 is located on chromosome 12q13 (OMIM *604653).
In 1964, Shahidi and colleagues described a brother and sister of French-Canadian descent who had hypochromic, microcytic anemia. These siblings also had elevated serum iron concentrations, massive deposition of iron in hepatocytes, and absence of stainable iron in the bone marrow. These children apparently had no defect in transferrin or heme synthesis. Two of their siblings appeared to have normal iron phenotypes. In 2004 and 2005, Priwitzerova and colleagues described a Czech female in a consanguineous kinship who came to medical attention at age 3 months because she had a syndrome of abnormal iron metabolism characterized by severe hypochromic, microcytic anemia, erythroid hyperplasia, abnormal erythroid maturation, elevated serum iron concentration, normal to slightly increased serum ferritin level, and markedly increased serum transferrin receptor levels. In 2005, Mims and colleagues reported that this woman was homozygous for a mutation in SLC11A2.
Clinical observations in patients with two SLC11A2 mutations are limited. In one case, left ventricular hypertrophy was detected before birth, and birth weight was low. Pallor is presumed to have been present in all reported cases.
Iron and the liver
The liver is the major site of iron storage in the body, and iron overload can cause hepatic fibrosis, cirrhosis, and hepatocellular carcinoma. (Table 5.1) In hereditary hemochromatosis, a pathologic expansion of body iron stores can occur due to excessive absorption of dietary iron (Chapters 2,8). The excess iron is preferentially deposited in parenchymal cells of the liver and other organs. When storage mechanisms are overwhelmed, iron in low-molecular weight forms can catalyze free radical reactions (Chapter 3). The resulting oxyradicals have the potential to damage cellular lipids, nucleic acids, proteins, and carbohydrates, resulting in wide-ranging impairment in hepatocyte function and integrity (Chapter 3). Damage can result in increased hepatic fibrogenesis, micronodular cirrhosis, and hepatocellular carcinoma. Important co-factors of iron-induced liver injury include chronic hepatitis C, excess alcohol consumption, and steatosis. Liver fibrogenesis shows a concordance with hepatic iron concentration and the duration of exposure to high iron levels. Phlebotomy therapy can reverse iron-induced hepatic fibrosis, but cirrhosis is less amenable to phlebotomy treatment.
In disorders of erythropoiesis, increased iron absorption and tissue iron deposition can occur. (Chapters 21–25). A common factor in iron-loading anemias is refractory anemia with a hypercellular bone marrow and ineffective erythropoiesis. These conditions include β-thalassemia, sideroblastic anemias, congenital dyserythropoietic anemias, and pyruvate kinase deficiency. In these syndromes, clinical and pathologic consequences similar to those seen in HFE hemochromatosis can occur.
In 1704 in Berlin, Heinrich Diesbach and Johann Konrad Dippel attempted to manufacture a synthetic red pigment. By accident, Dippel mixed potash, animal oil derived from blood, and iron sulfate. Thereafter, he discovered that he had produced an insoluble, light-fast, dark blue pigment. This color was first used extensively to dye the uniforms of the Prussian army, and became known as “Prussian blue.” Almost 150 years later, physicians and scientists recognized the feasibility of visualizing iron in tissue using a similar staining sequence. After more than 250 years, it became practical to quantify iron in blood and tissue, permitting case finding and screening for hemochromatosis and iron overload. Maneuvers to treat iron overload began in the same era. In the interval 1994–1996, the genetic bases of four different iron disorders (X-linked sideroblastic anemia, aceruloplasminemia, hereditary hyperferritinemia-cataract syndrome, and HFE hemochromatosis) were elucidated. The pace of basic science, clinical, and sociological revelations pertinent to hemochromatosis and iron overload disorders continues to accelerate. This chapter provides an abbreviated chronology of these discoveries.
Iron in tissue
In 1847, Rudolph Virchow reported the occurrence of golden brown granular pigment at sites of hemorrhage and congestion in tissue examined by microscopy. The pigment was soluble in sulfuric acid, yielded a red ash on ignition, and produced a positive Prussian blue reaction. In 1867, Max Perls formulated the first practical acidified ferrocyanide reaction for histologic analysis of iron, and applied the staining reaction to a variety of tissues.
Hepcidin, an antimicrobial peptide produced by hepatocytes, is a central negative regulator of iron absorption that is encoded by the HAMP gene on chromosome 19q13 (Chapter 2). In humans, HAMP mutations account for a rare subtype of juvenile-onset hemochromatosis (OMIM #602390). Some patients have an autosomal recessive disorder associated with homozygosity for rare pathogenic HAMP mutations. Others have hemochromatosis phenotypes due to heterozygosity for a pathogenic HAMP mutation and co-inheritance of heterozygosity or homozygosity for HFE C282Y.
The precursor of hepcidin comprises 84 amino acids, from which 3 active peptides of 25, 22, and 20 amino acids, respectively, are produced by protease cleavage. The 25 and 20 amino acid peptides represent the major forms. Active forms of hepcidin contain numerous cysteines. Eight highly-conserved cysteine residues form four disulfide bonds, the critical basis of a rigid structure of the final peptide. The HAMP promoter contains consensus sequences for the transcription factor CCAAT/enhancer binding protein-α (CEBP/α) that confers liver tissue specificity. The HAMP promoter also responds to interleukin-6 (IL-6), and has a bone morphogenetic protein-responsive element (BMP-RE) that binds SMAD 1/5/8/4 protein complex. Hepcidin expression is decreased in HFE, “gain-of-function” SLC40A1, and TFR2 hemochromatosis, and increased in “loss-of-function” SLC40A1 hemochromatosis in the absence of HAMP mutations (Chapters 8, 12, 15). In experimental animals, hepcidin synthesis is increased by iron loading and inflammation and is inhibited by iron deficiency anemia and hypoxia.
Clinical and laboratory features
Patients who are homozygous for deleterious HAMP mutations have clinical phenotypes similar to those of patients with HJV hemochromatosis.
Scientific and clinical questions are usually answered partially and in increments. Most of the questions posed by Joseph Sheldon in 1935 and at the First International Conference on Hemochromatosis in 1987 have been answered. Since the discovery of HFE in 1996, there has been an explosion of research interest and reports related to iron biology and diseases of iron homeostasis. Some important “unknowns” presented in the conclusion of a major 2000 text devoted to hemochromatosis have been resolved. This section presents some of the important old and new questions for which answers are needed and changes in medical care delivery are predicted. These topics of interest include the biology and genetics of iron homeostasis and iron overload, hemochromatosis and iron overload screening, advances in diagnosis, complications of iron overload, and improvements in management.
Biology and genetics of iron homeostasis and iron overload
It is assumed that the common HFE mutations C282Y and H63D conferred some evolutionary advantage, but the mechanism(s) by which such a putative advantage(s) was mediated is not known. C282Y, found predominantly in western European Caucasians, is the most common known mutation that has a profound effect on iron homeostasis and phenotype. Many have inferred that this polymorphism fostered an iron procurement advantage for women during their reproductive years, although this is unproven. Related possibilities include the conjecture that C282Y either increases fertility of women (or men), or promotes greater survival of fetuses in utero or of newborns.
Mutations in the SLC40A1 (FPN1) gene that encodes ferroportin (OMIM *604653) cause an uncommon, heterogeneous group of iron overload disorders characterized by an autosomal dominant pattern of inheritance (OMIM #606069). Ferroportin hemochromatosis has been described worldwide in a variety of race/ethnicity groups. SLC40A1 mutations cause two major iron overload phenotype patterns, each depending on the particular mutation and its effect on the function of the transcribed ferroportin protein. In many ferroportin hemochromatosis kinships, serum iron measures and complications of iron overload typical of other types of hemochromatosis are relatively uncommon. The collective term “ferroportin disease” or “hemochromatosis type 4” is sometimes used to describe the clinical manifestations of ferroportin mutations.
In 1990, an autosomal dominant form of hemochromatosis was reported in a Melanesian pedigree from the Solomon Islands. All affected individuals had serum iron measures and a pattern of liver iron staining similar to those of HFE hemochromatosis, although linkage of this disorder to chromosome 6p was excluded. In 1999, Pietrangelo and colleagues reported a large Italian family that included persons with an iron overload condition that occurred in pattern consistent with autosomal dominant inheritance. Based on microsatellite marker analyses, this disorder was also not linked to chromosome 6p. In 2001, Njajou and colleagues identified the SLC40A1 mutation N144H associated with autosomal dominant hemochromatosis in a large multi-generation family from the Netherlands.
X-linked sideroblastic anemias (XLSA) are characterized by impaired mitochondrial iron metabolism, “ringed” sideroblasts and increased erythropoiesis. In some cases, these disorders cause parenchymal iron overload similar to that of hemochromatosis. Increased iron absorption is upregulated by relative or absolute suppression of hepcidin expression presumably mediated by ineffective erythropoiesis through growth/differentiation factor 15 (GDF15). Iron overload in some patients is exacerbated by erythrocyte transfusion. Mutations in the ALAS2 gene that encodes erythroid-specific 5-aminolevulinate synthase (ALA synthase) account for most cases (OMIM #300751), although rare mutations in other genes on the X chromosome or elsewhere also cause sideroblastic anemia phenotypes and variable degrees of iron loading. Anemia in two-thirds of patients with ALAS2 mutations can be alleviated with simple, non-transfusion therapy. Early recognition and treatment of iron accumulation prevents irreversible organ damage. Informal experience suggests that XLSA may be more common than is generally recognized.
ALAS2 sideroblastic anemia
In 1945, Thomas Cooley described the first cases of XLSA in two brothers from a large family in which the inheritance of the condition was documented through six generations. Additional observations in this family and description of a new kinship were published by Rundles and Falls in 1946. In 1956, Harris and co-workers reported the first case of “pyridoxine-responsive anemia.” Ringed sideroblasts were described by Bjorkman in 1956 (Fig. 25.1). Byrd and Cooper coined the term “hereditary iron-loading anemia” in 1961. The term “sideroblastic anemia” was adopted in 1965. By this time, the association of anemia sometimes responsive to pyridoxine and iron overload were widely recognized.
Acquired sideroblastic anemia is a heterogeneous group of disorders characterized by excessive accumulation of amorphous iron deposits in the mitochondria of erythrocyte precursors. Ring sideroblasts, the sine qua non of sideroblastic anemia, are erythroblasts in Prussian blue-stained marrow aspirates that have numerous iron-positive mitochondria that occur in a perinuclear distribution. In sideroblastic anemia, typical ring sideroblasts have a full or partial ring and usually comprise more than 5% of all erythroblasts (Chapter 25, Fig. 25.1a, b). Other erythroblasts in the same specimens may have markedly increased numbers of iron-positive mitochondria that do not form a perinuclear halo. Acquired sideroblastic anemias encompass two main categories: clonal hematopoietic stem cell disorders classified as myelodysplastic syndromes (MDS); and reversible types of sideroblastic anemia due to drugs, chemicals, or other factors. Heritable types of sideroblastic anemia are described in Chapter 25.
Acquired sideroblastic anemia due to myelodysplastic syndromes
Refractory anemia with ring sideroblasts (RARS) is characterized by erythroid dysplasia, mitochondrial accumulation of mitochondrial iron-containing ferritin (mitoferrin), defective erythroid maturation, and anemia; some patients also have elevated platelet counts. Other patients with MDS and ring sideroblasts are classified as having refractory cytopenias with multilineage dysplasia and ring sideroblasts (RCMD-RS). MDS in patients with either RARS or RCMD-RS may progress to excess marrow blasts (refractory anemia with excess blasts, RAEB) or acute leukemia.
Non-germline mutations in marrow stem cells underlie the pathogenesis of MDS, including MDS subcategories typically associated with ring sideroblasts.
In 1922, German investigators Hallervorden and Spatz reported a syndrome of neurologic and pathologic findings in a sibship of 12 individuals, among whom 5 siblings had progressive dysarthria and dementia. At autopsy, the investigators observed brown discoloration of the substantia nigra and the globus pallidus of the affected siblings. Since the initial report, hundreds of individuals with this disorder have been reported, and most have mutations of the PANK2 gene that encodes pantothenate kinase 2. In 2001, this disorder was named pantothenate kinase-associated neurodegeneration, also known as neurodegeneration with brain iron accumulation (OMIM #234200). The brown discoloration of the brains of persons with pantothenate kinase-associated neurodegeneration is caused by the deposition of excessive quantities of iron. This condition occurs in approximately 3 per 1,000,000 people. In a university hospital autopsy series that was evaluated to identify iron overload disorders specifically, only one case of neurodegeneration with brain iron accumulation was found in 10,345 adults (age ≥21 years) and 1337 children (>1 year of age).
There is phenotypic heterogeneity in the clinical presentation of patients who have mutations of the PANK2 gene. The report of an international study published in 2003 describes findings in 186 patients from 145 families, including clinical histories, physical examination findings, laboratory characteristics, extrapyramidal neurologic abnormalities, and magnetic resonance imaging evidence of iron deposition in the basal ganglia.
Iron is an essential element, but in excess it can result in cell injury (Table 3.1). When storage mechanisms are overwhelmed, iron in low molecular weight forms can play a catalytic role in the initiation of free radical reactions. The resulting oxyradicals have the potential to damage cellular lipids, nucleic acids, proteins, and carbohydrates, resulting in wide-ranging impairment in cellular function and integrity. The rate of free radical production must overwhelm the cytoprotective defenses of cells before injury occurs.
In HFE hemochromatosis, there can be a pathologic expansion of body iron stores due to an increase in the absorption of dietary iron. Transferrin saturation is increased and non-transferrin-bound iron (which is redox-active) may be present. The excess iron is preferentially deposited in the cytoplasm of parenchymal cells of various organs and tissues including the liver, 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 involved organs and include liver failure (from cirrhosis), diabetes mellitus, arthritis, cardiac dysfunction (arrhythmias and failure), and hypogonadotrophic hypogonadism. Important co-factors of iron-induced liver injury include chronic hepatitis C and excess alcohol consumption. Although cadmium and lead may also be transported by divalent metal transporter-1, the major apical iron transporter in enterocytes, excess iron is considered to be the major cause of toxicity in hemochromatosis.
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