PRESIDENT’S SYMPOSIUM FROM

THE

2013 SOUTHERN REGIONAL MEETING

Hemochromatosis and Iron Overload: From Bench to Clinic James C. Barton, MD

Key Indexing Terms: Arthropathy; Cirrhosis; Diabetes; Hemochromatosis; HFE; Hepcidin; Iron. [Am J Med Sci 2013;346(5):403–412.]

he first studies of iron in human tissues were published more than 160 years ago and those of iron overload more than 140 years ago. Through the early 20th century, few scientists and physicians explored this then unusual area of inquiry. Consequently, discoveries were infrequent and often fortuitous. In the past 60 years, many basic science and clinical investigators characterized the physiology of iron absorption and metabolism and the heritable nature and clinical manifestations of hemochromatosis. During the past 20 years, scientists defined the genetic and molecular basis of iron homeostasis. Clinicians diagnosed patients and identified research participants whose genetic and phenotypic diversity germane to iron-related disorders far exceeded most earlier speculations. Herein, selected historical, scientific and clinical topics about hemochromatosis and iron overload are succinctly reviewed. Some unanswered basic research and clinical questions are posed in conclusion.

T

DEMONSTRATION OF NON-HEME IRON IN TISSUE In 1704 in Berlin, Heinrich Diesbach, a colormaker and painter, and Johann Konrad Dippel, an alchemist and physician, attempted to synthesize a red pigment. Dippel accidentally mixed potash and animal oil derived from blood with iron sulfate and produced an insoluble, light-fast, dark blue pigment (Berliner blau).1 This color reaction was used to dye the uniforms of the Prussian army and thus the pigment became widely known as “Prussian blue.” 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 produced a positive Prussian blue reaction.2 In 1867, Perls formulated the first practical acidified ferrocyanide reaction for histological analysis of non-heme iron and applied the staining reaction to a variety of tissues.3 In 1962, Scheuer et al reported a method of grading iron stained using Perls’ technique in hepatic biopsy specimens from patients with hemochromatosis and their relatives and described the characteristic gradient of iron distribution in hepatic lobules in hemochromatosis.4

From the Southern Iron Disorders Center, Birmingham, Alabama and Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama. Supported in part by the Southern Society of Clinical Investigation and by Southern Iron Disorders Center. Presented as part of the Southern Society for Clinical Investigation’s Presidential Symposium at the Southern Regional Meeting, February 22, 2013, New Orleans, Louisiana. The authors have no other conflicts of interest to disclose. Correspondence: James C. Barton, MD, Southern Iron Disorders Center, 2022 Brookwood Medical Center Drive, Birmingham, AL 35209 (E-mail: [email protected]).

The American Journal of the Medical Sciences



EARLY REPORTS OF HEMOCHROMATOSIS Trousseau reported the syndrome of hepatic cirrhosis, pancreatic fibrosis and cutaneous hyperpigmentation in 1865, but he did not recognize the involvement of iron in its pathogenesis.5 Troisier’s account of “diabète bronzé et cirrhose pigmentaire” in 1871 confirmed and extended that of Trousseau and reported the presence of iron-reactive pigment in various tissues.6 Troisier’s syndromic triad of diabetes, skin hyperpigmentation and cirrhosis became the sine qua non of hemochromatosis diagnosis for decades. In 1889, von Recklinghausen used methods of Virchow and Perls to detect excess iron in tissues obtained at autopsy of 12 persons who had “hämochromatose.”7 He believed that the ironcontaining pigment was derived from blood (due to hemorrhage or hemolysis). French clinicians in the early 1930s reported “le syndrome endocrine-hepato-cardiaque.”8,9 This latter group of disorders was later called “juvenile hemochromatosis.” In 1935, Sheldon, an English gerontologist, summarized 311 carefully selected “haemochromatosis” cases gleaned from the literature.10 He concluded that the absorption of iron (and possibly that of other metals) is increased in hemochromatosis and suggested that the disorder is an “inborn error of metabolism” that primarily affects men. Sheldon rejected hypotheses that diabetes, infections, intoxication, alcoholism and other conditions cause hemochromatosis. As late as the 1960s, some pathologists opined that iron overload and tissue injury in Caucasians with hemochromatosis are consequences of alcoholism and other nutritional factors.11 Clinicians published increasing evidence that Caucasians with hemochromatosis had a heritable trait that increased iron absorption and recognized that the clinical phenotype of hemochromatosis was influenced by age, sex and other attributes.

HERITABILITY OF HEMOCHROMATOSIS In 1975, Simon et al in Brittany reported that a genetic factor associated with hemochromatosis phenotypes was closely linked to the human leukocyte antigen (HLA)-A*03 locus on chromosome 6p.12 HLA-A*03 was also linked to HLA-B*07 or B*14 in many French hemochromatosis kinships. Inheritance patterns of HLA haplotypes confirmed that hemochromatosis phenotypes are manifest as autosomal recessive traits. By the late 1970s, relatives of hemochromatosis probands who also inherited 2 HLAlinked hemochromatosis alleles were identified with HLA-A and HLA-B immunophenotyping, sometimes before they developed iron overload. In 1979, Utah investigators described the use of serum and hepatic iron measures as diagnostic criteria in index patients with hemochromatosis and their relatives.13 In 1988, Edwards et al reported the results of a study in which approximately 11,000 white Utah blood donors were screened for hemochromatosis using iron phenotyping and HLA-A and HLA-B typing.14 They demonstrated that hemochromatosis is a common autosomal recessive disorder linked to HLA on chromosome 6p and confirmed that its penetrance is greater in men than women. In the interval 1994 to 1996, Maria de Sousa et al in Portugal reported that mice with heritable deficiency of b2microglobulin (beta 2m2/2) have iron phenotypes in the intestine, blood and liver that are similar to those of persons with hemochromatosis. They also concluded that a b2-microglobulindependent gene product is involved in iron homeostasis.15,16

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HFE AND HEPCIDIN In 1996, Feder et al used a positional cloning technique to evaluate DNA specimens from a cohort of whites with hemochromatosis phenotypes.17 They discovered an atypical major histocompatibility region class I gene on chromosome 6p. In almost every case, the gene, later named HFE (high iron), contained missense mutations. Most hemochromatosis subjects studied by them had homozygosity for the C282Y mutation (exon 4: 845G/A). A few subjects had the mutation H63D (exon 2: 187C/G) that occurred in the genotypes C282Y/ H63D or H63D/H63D.17 HFE protein was the b2-microglobulin-dependent gene product predicted by de Sousa et al.15,16 Hepcidin is a low–molecular weight protein with many cysteine pairs described independently in 2000 to 2001 by Krause, Ganz and Pigeon.18–20 The hepcidin gene HAMP (hepatic antimicrobial peptide) is highly conserved. Hepcidin is synthesized by the liver, is present in the plasma, and is excreted in the urine. Hepcidin is the central regulator of iron absorption. The production or action of hepcidin is decreased in all hemochromatosis syndromes.21 Increased levels of hepcidin account for the ferrokinetic abnormalities typical of the anemia of chronic disease.21 Expression of normal HFE protein is greatest in cells in which iron traffic is high. These include duodenal crypt cells, hepatocytes, tissue macrophages, circulating monocytes and syncytiotrophoblasts but not erythroid cells.22,23 b2-microglobulin binds to the extracellular domain of wild-type HFE.17 Transferrin receptor (TFR1) and transferrin receptor 2 (TFR2) bind HFE in a pH-dependent manner. HFE competes with diferric transferrin for binding to TFR1. HFE expression is decreased in hemochromatosis due to protein abnormalities consequent to HFE mutations.23 Dissolution of cysteine-dependent disulfide bonds in HFEC282Y abrogates b2-microglobulin binding.24 Mutant HFE protein impairs bone morphogenic protein signaling and prevents sufficient synthesis of hepcidin.25 To date, no immune function of HFE has been substantiated.

HFE HEMOCHROMATOSIS IN THE 21ST CENTURY

After the discovery of HFE in 1996,17 it was confirmed that C282Y homozygosity was the predominant HFE genotype of western European whites with hemochromatosis and that the C282Y allele was tightly linked with HLA-A*03 in many western Europeans.26 The frequencies of HFE C282Y and C282Y homozygosity are greatest in northwestern European populations. In Europe, C282Y is most prevalent in the British Isles (especially Ireland), Norway and Brittany. Spread largely by

TABLE 1. HFE C282Y homozygotes in general populations Population/ethnicity Size of cohort, n Irish adults Norwegian adults Australian white adults Alabama white adults Brittany newborns Southern California white adults North American white adults Hispanic adults African American adults Asian adults

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404 65,238 3011; 31,192 9316 1000 30,418 44,082 12,459 27,124 12,272

Vikings, C282Y occurs in a decreasing frequency gradient in more eastern and southern Europe and the Near East. Outside Europe, the frequency of C282Y is relatively high in whites in Alabama and Australia. This is due to the high proportion of British Isles ancestry among whites who migrated to these geographic areas. The prevalence of C282Y homozygosity is much lower in non-European populations in which the occurrence of C282Y is attributed to admixture with white populations (Table 1). The HFE H63D allele is cosmopolitan although its frequency in western European whites is relatively high.36 In the early 21st century, HFE hemochromatosis emerged as the most common, potentially consequential autosomal recessive disorder of European whites. Approximately 90% of white patients with hemochromatosis phenotypes have C282Y homozygosity. Due in part to abnormal HFE protein function in HFE C282Y homozygotes, hepcidin expression is inappropriately low for the rate of iron absorption and the magnitude of body iron stores.37 C282Y homozygotes have increased risk to develop iron overload and consequent damage to liver, pancreas, heart, joints and other tissues, although organ-specific complications vary among patients diagnosed in nonscreening venues. Phenotype and genotype characteristics of HFE hemochromatosis are displayed in Table 2. In office practices, the most common reasons for referral of patients subsequently diagnosed to have HFE hemochromatosis are elevation of serum concentrations of hepatic transaminases (usually mild), hyperferritinemia and occurrence of diabetes, arthropathy or hypogonadism.39 Most patients are in their 4th to 6th decades of life. The ratio of men to women with hemochromatosis in practice settings is approximately 10:7. Weakness and fatigue, abdominal discomfort, arthralgias and decreased libido and erectile dysfunction were once typical symptoms. In contrast, many patients diagnosed with hemochromatosis in nonscreening venues today are asymptomatic because their hemochromatosis is first suspected due to biochemical abnormalities alone. Physical examination may reveal evidence of arthropathy (especially in hands, hips and knees), stigmata of chronic liver disease or cutaneous hyperpigmentation, although there are no pertinent physical abnormalities in many patients.39 In the North American Hemochromatosis and Iron Overload Screening (HEIRS) Study,40 previously and newly diagnosed C282Y homozygotes with elevated serum ferritin levels had higher prevalences of chronic fatigue and had more hyperpigmentation and swelling or tenderness of the 2nd and 3rd metacarpophalangeal joints on physical examination than control subjects.41 Reports of joint stiffness were also more prevalent in newly diagnosed C282Y homozygotes with elevated serum

Homozygotes per 1000

Reference

12.4 6.8 5.0; 6.8 6.1 5.0 4.6 4.4 0.27 0.14 0.00039

27 28 29,30 31 32 33 34,35 34 35 34

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TABLE 2. Characteristics of HFE hemochromatosis Iron overload phenotype HFE genotype Uncommon Increased iron absorption by enterocytes Serum iron, transferrin saturation increased due to increased iron release by macrophagesa Increased storage ironc

Common Usually C282Y/C282Y Sometimes C282Y/H63D or C282Y/S65C (iron phenotypes usually mild)b .60 pathogenic HFE alleles, most rare; most occur in trans heterozygosity with C282Y Rare patients have other ironrelated mutationse

Hepcidin expression inappropriately low for body iron storesd Common non-iron liver disorders H63D/H63D rarely causes iron may mimic iron overload overloadb f phenotypes a

May occur in the absence of increased iron storage. Most patients with these genotypes and hemochromatosis phenotypes have non-iron liver disease. c Liver is the predominant target organ for iron overload. Serum ferritin (surrogate test), hepatic iron concentration or iron removed by phlebotomy to achieve iron depletion (“quantitative” phlebotomy). MRI with T2* technique and superconducting quantum interference device methods are less practical. d Low hepcidin in serum and urine; hepcidin measures usually not available nor needed in nonresearch settings. e For example, HAMP alleles (“digenic” hemochromatosis).38 f Alcoholic liver disease, non-alcoholic fatty liver disease, chronic viral hepatitis C. b

ferritin levels than among control subjects. The sex- and ageadjusted prevalences of self-reported symptoms and signs of liver disease, heart disease, diabetes and most other manifestations were similar in previously undiagnosed C282Y homozygotes and control subjects at the time of their respective enrollments in the HEIRS Study.41 Laboratory evaluation detects elevated serum iron and transferrin saturation values, slightly low total serum ironbinding capacity, hyperferritinemia and elevated serum concentration of hepatic transaminases in many cases. Commercially available HFE mutation-specific analyses (C282Y, H63D, S65C alleles) confirm that most white patients with hemochromatosis phenotypes and iron overload are C282Y homozygotes. Percutaneous liver biopsy is recommended in patients whose serum ferritin at diagnosis is .1000 mg/L.42 Routine histology (hematoxylin and eosin technique), Perls’ Prussian blue staining, Mallory’s trichrome stain to demonstrate fibrosis and hepatic iron concentration should be evaluated on each liver biopsy specimen. Hepcidin measurements are not indicated. Patients with abdominal pain, non-iron liver conditions, diabetes mellitus, arthropathy, or hypogonadism require further evaluation. In population and family screening studies, many C282Y homozygotes have body iron burdens that are low, normal or only slightly elevated.33 Iron overload may not progress in such individuals even if they are not treated with phlebotomy, although annual monitoring of serum ferritin is recommended.42 Thus, HFE homozygosity alone in many patients is insufficient to cause iron overload. It is assumed that non-HFE factors, both acquired and heritable, account for the development of iron overload in some but not all C282Y homozygotes and would Ó 2013 Lippincott Williams & Wilkins

explain the heterogeneity of disease expression observed in HFE hemochromatosis case series.

TISSUE AND ORGAN INJURY IN IRON OVERLOAD Excess iron is normally stored in ferritin molecules. Synthesis of H- and L-ferritin mRNAs is regulated by a 59-UTR iron-specific stem-loop structure (iron-responsive element). Smaller quantities of iron are bound to transferrin, but transferrin synthesis is not up-regulated when body iron content is increased. In iron overload, non-heme iron unincorporated into ferritin and unbound to transferrin (eg, “non-transferrin-bound iron”) may cause tissue injury with diverse consequences. Two major (but not mutually exclusive) mechanisms have been proposed to describe the toxicity induced by iron overload.43 The oxidative injury hypothesis postulates that iron overload in vivo results in the formation of oxyradicals that damage cellular constituents including lipids, nucleic acids, proteins and carbohydrates and impair calcium homeostasis and cellular function. The lysosomal injury hypothesis proposes that excessive accumulation of iron within lysosomes can lead to lysosomal fragility, impaired lysosomal function and eventual cellular injury through the release of hydrolytic enzymes and stored iron into the cytoplasm. In addition, iron-induced oxyradicals may damage hepatic mitochondria, endoplasmic reticulum, plasma membranes and DNA.43

LIVER The liver, a major site of iron storage in healthy persons, is the principal target of iron overload in hemochromatosis. In healthy adults, iron occurs in relatively small amounts in both hepatocytes and Kupffer cells. In HFE hemochromatosis, iron is deposited preferentially in hepatocytes, and Perls’ Prussian blue staining demonstrates that intrahepatocytic iron occurs in a decreasing gradient from periportal to centrilobular areas of hepatic lobules. With severe iron overload, increased amounts of iron also appear in bile ductular cells and hepatic macrophages (Kupffer cells). The major causes of death in persons with hemochromatosis are cirrhosis and its complications, including primary liver cancer. Iron-induced activation of stellate cells in the liver may induce fibrogenesis and eventually cause cirrhosis. Approximately 15% patients with HFE hemochromatosis have other liver conditions, including alcoholic liver disease, non-alcoholic fatty liver disease and chronic viral hepatitis. These conditions may act in synergy with hepatocellular iron overload to increase liver injury, iron deposition and cirrhosis risk. Iron is also a hepatic carcinogen. The incidence rate of primary liver cancer in persons with hemochromatosis is high, especially in those with cirrhosis.44,45 Mechanisms whereby iron may be involved in carcinogenesis include induction of oxidative damage of DNA, facilitation of tumor proliferation and modifications of the immune system.46 The quantity of iron deposited in the liver is the major known predictor of cirrhosis in hemochromatosis. Serum ferritin .1000 mg/L at diagnosis, male sex and diabetes typically occurred in the same individuals in older hemochromatosis case series. In more recent cohorts of C282Y homozygotes diagnosed in settings other than general population screening, cirrhosis occurs in some individuals at all levels of iron overload. Other patients with severe iron overload do not have cirrhosis on specimens of liver obtained by biopsy. Regression analyses of multiple independent variables of known pertinence indicate that only 20% to 35% of cirrhosis risk in non-screening C282Y homozygotes is associated with iron overload severity.

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The other 65% to 80% of putative cirrhosis risk is attributed to genetic and acquired factors that remain to be delineated.

PANCREAS The diagnostic triad of hyperpigmentation, diabetes mellitus and cirrhosis was proposed in the 19th century.4–7 Reports from the early 20th century demonstrated that approximately 80% of patients with hemochromatosis had diabetes, typically associated with cirrhosis and severe hemosiderin deposition in pancreatic acini and cells of the islets of Langerhans.10,48,49 Specificity of iron deposition for the beta cells of the islets was reported in 1987.50 In 1968, Balcerzak et al51 reported that the prevalence of diabetes in families of patients with hemochromatosis was high, and diabetes was associated with elevated levels of circulating insulin. In 1972, Dymock et al52 reported that 25% of hemochromatosis patients with diabetes had first-degree relatives who also had diabetes, whereas only 4% of hemochromatosis patients without diabetes had a first-degree relative with diabetes. The reports of Balcerzak and Dymock thus suggested that a non-iron heritable factor was a prominent cause of diabetes in persons with hemochromatosis. The diagnosis of hemochromatosis changed in 1996 after the discovery of the HFE gene and the C282Y polymorphism.17 More persons were diagnosed to have hemochromatosis using a genetic criterion (C282Y homozygosity) rather than iron phenotyping alone. In hemochromatosis case series from the early 21st century, the prevalence of diabetes in persons with hemochromatosis phenotypes was much lower than typically reported in the 20th century.53,54 In a 2008 report, neither HFE genotype, serum ferritin level at diagnosis, nor cirrhosis predicted the development of diabetes in persons with hemochromatosis phenotypes.54 In the HEIRS Study, prevalences of self-reported diabetes in adult C282Y homozygotes and in participants without HFE C282Y or H63D did not differ significantly.41 Taken together, these observations suggest that diabetes in patients diagnosed with hemochromatosis in the early part of the 21st century or later is less prevalent than in earlier hemochromatosis cases series, is infrequently caused by iron overload and may be due predominantly to non-HFE familial traits typical of type 2 diabetes in persons without hemochromatosis diagnoses.

JOINTS

Hemochromatosis arthropathy was first described in 1964 by Schumacher.55 Many patients with severe characteristic arthropathy have severe iron overload and consequent extra-articular manifestations of iron overload. Arthropathy typically affects the 2nd and 3rd metacarpophalangeal and proximal interphalangeal joints, hips and knees. Other diarthrodial joints are affected in some patients. Arthropathic changes include hypertrophy, subchondral cysts at metacarpal heads, narrowed joint spaces, chondrocalcinosis and “hook” osteophytes.39 Prussian blue staining demonstrates iron deposition in cartilage and synovial type B cells, although a similar pattern of iron deposition has been observed in other types of arthropathy. Arthropathy is a predominant cause of disability among persons with hemochromatosis diagnosed in nonscreening venues. A similar type of hand arthropathy occurs in HFE C282Y heterozygotes.56 In the HEIRS Study, previously diagnosed C282Y homozygotes and newly diagnosed homozygotes in North America with elevated serum ferritin levels had higher prevalences of swelling or tenderness of the 2nd and 3rd metacarpophalangeal joints on physical examination than control subjects. Joint stiffness was also more common among newly

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diagnosed C282Y homozygotes with elevated serum ferritin than among control subjects.41

TREATMENT

Phlebotomy to achieve iron depletion is the “gold standard” of therapy for iron overload due to HFE and other types of hemochromatosis.42 Fundamental management recommendations are displayed in Table 3. Details of treatment and outcome expectations for HFE hemochromatosis and its complications are presented in detail elsewhere.42

HEMOGLOBIN AND MEAN CORPUSCULAR VOLUME In untreated HFE C282Y homozygotes, the mean hemoglobin concentration is greater by approximately 1.0 g/dL than that of control subjects without common HFE mutations.57,58 The mean corpuscular volume (MCV) of untreated C282Y homozygotes is also greater, sometimes above the upper reference limit, even in patients without liver disease or delayed nuclear maturation of erythroid cells.57 The mean red blood cell count and mean coefficient of variation of MCV (red blood cell distribution width, RDW) are lower in C282Y homozygotes.57 These characteristics, usually more pronounced in men than women, are due to the greater amounts of iron available to marrow erythroblasts via transferrin, the iron saturation of which is increased in most C282Y homozygotes. Genome-wide linkage analyses confirmed that a locus (loci) on chromosome

TABLE 3. Management of hemochromatosis Physician Patient Phlebotomy removes ;200 mg Fe/unita Treat when serum ferritin .300 mg/L men, .200 mg/L women Achieve iron depletion (serum ferritin ,50 mg/L)b Maintain serum ferritin ,300 mg/L men, ,200 mg/L women Manage liver disease, diabetes, arthropathy, hypogonadism as for patients without hemochromatosis Screen for primary liver cancer in patients with cirrhosisc Iron chelation therapy rarely indicatede

No dietary iron content manipulation indicated Consume alcohol in moderation; if cirrhosis, abstain from alcohol No uncooked shellfish; avoid seawater drippings No supplemental iron; limit vitamin C to 500 mg daily Maintain optimal weight; control blood glucose levels

Tannate in tea binds inorganic iron, blocks absorptiond Protein pump inhibitors decrease frequency of maintenance phlebotomyd

One unit of blood 5 430 to 500 mL. After diagnosis, serum iron and transferrin saturation levels are not informative. Monitor iron depletion therapy with serum ferritin alone. c Optimal method has not been determined. Consider annual imaging with ultrasonography or CT scanning or with change in status. Serum alpha-fetoprotein levels are elevated in ;20% of persons with primary liver cancer. d Coincidental use may benefit some patients in the maintenance phase of phlebotomy treatment but will not remove stored iron. Prescribe and use proton pump inhibitors for licensed indications only. e Some patients with “juvenile” hemochromatosis who present with cardiomyopathy due to cardiac siderosis may benefit from adding iron chelation treatment to aggressive phlebotomy. Iron-induced cardiomyopathy is very rare in HFE hemochromatosis. a b

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6p21 is significantly associated with hemoglobin concentration.59 This locus is presumed to be HFE.

ABSORPTION OF NON-FERROUS METALS The absorption or retention of some non-ferrous divalent metals is increased in persons with hemochromatosis homozygosity.39 Absorption and retention of the same metals are also increased in hemochromatosis heterozygotes, but to a lesser degree.39,60 On the other hand, the absolute numbers of heterozygotes far exceed those of C282Y homozygotes.39 Increased absorption of non-ferrous divalent metals has implications for nutrition, toxicology and occupational and environmental radiochemical exposure. Divalent metal transporter-1 (DMT-1), expressed on the luminal surfaces of absorptive enterocytes, is the major Fe2+ transporter that mediates cellular iron uptake. DMT-1 is up-regulated in hemochromatosis, in iron deficiency and after phlebotomy. DMT-1 binds Fe2+, Zn2+, Mn2+, Co2+, Cd2+, Cu2+, Ni2+ and Pb2+.61 In hemochromatosis homozygotes, the absorption of Co2+, Cd2+ and Pb2+ is 1.5 to 3.0 times greater than in normal control subjects.60,62,63 Hepatic concentrations of zinc, manganese and copper are several-fold greater in hemochromatosis homozygotes than in normal subjects.64,65 Some non-ferrous metals that bind DMT-1 may also be exported from enterocytes by ferroportin, although this is unproven. Some divalent cations, especially Zn2+, Cd2+, Cu2+ and Pb2+, are also absorbed and exported into the blood via mechanisms uninvolved with iron.

IMMUNOGLOBULINS Subnormal serum levels of immunoglobulin G subclass 3 (IgG3) occur in approximately 30% of HFE C282Y homozygotes diagnosed in nonscreening venues. Some homozygotes also have subnormal IgG1 levels. Most have normal levels of IgA or IgM.66 In C282Y homozygotes, IgG subclass deficiency is significantly associated with the HLA haplotypes A*03, B*07 and A*01, B*08. In kinships, IgG subclass deficiency segregates with HLA-A and HLA-B haplotypes.66 These observations indicate that a gene that suppresses serum IgG levels is linked to HFE C282Y on chromosome 6p, although the precise locus and mechanism of action of this putative allele(s) have not been determined. Iron overload severity at diagnosis and achieving iron depletion with phlebotomy are not significantly related to serum IgG subclass levels. Some hemochromatosis patients with selective IgG subclass deficiency, especially those with IgG1/IgG3 deficiency, have recurrent upper and lower respiratory tract infections and benefit from IgG monthly replacement therapy.66

VIBRIO VULNIFICUS AND OTHER BACTERIAL PATHOGENS

Vibrio vulnificus (5volnificus; “wound former”), a temperature-dependent Gram-negative bacterium described in 1979, occurs worldwide in temperate coastal waters.67 V vulnificus is virulent and highly dependent on iron.68 Typical infections are characterized by septicemia, fever and painful hemorrhagic cellulitis of the extremities that occur within a few days of consumption of raw shellfish (usually oysters, clams or mussels) or other food items contaminated with seawater drippings.69,70 V vulnificus can also enter tissues through minor wounds and, within a few hours, cause a clinical picture similar to that associated with ingestion of V vulnificus. The recommended antibiotic therapy for V vulnificus infection is doxycycline, 100 mg intravenously or orally twice daily, and ceftazidime, 2 g intravenously every 8 hours. Cefotaxime or ciprofloxacin is also effective. Most patients with V vulnificus infections require much in-hospital care, including Ó 2013 Lippincott Williams & Wilkins

excision of necrotic tissue. The death rate is approximately 50% in persons with septicemia and approximately 15% in those with wound infections. In the United States, V vulnificus infections are the leading cause of death related to seafood consumption. Most infections emanate from the Gulf of Mexico and occur in the summer and early fall.69,71 The incidence of infections is greater in men than women. Of persons who develop endotoxic shock, approximately 85% are men. Many infections occur in persons diagnosed to have hemochromatosis, cirrhosis and other liver diseases of non-iron cause, depressed immunity or impaired renal function.68,70,71 In persons with hemochromatosis, both septicemia and wound infections have been reported.68,72 The greater iron burden of many men with hemochromatosis and their relatively low hepcidin levels may increase infection risk. Patients with hemochromatosis should be advised to avoid consumption of uncooked shellfish, regardless of iron or liver status.42 Cooking destroys V vulnificus, making shellfish consumption safe. Primary peritonitis, septicemia or meningitis due to Escherichia coli has been described in several adults with hemochromatosis, some of whom were suspected or diagnosed to have cirrhosis.73–75 Like V vulnificus, E coli requires iron although it is unknown whether persons with hemochromatosis have greater susceptibility to infection with E coli than persons without hemochromatosis diagnoses.

EVOLUTIONARY ADVANTAGE OF HFE C282Y Population genetic analyses suggest that HFE C282Y arose in the Neolithic Age, more than 4000 years ago.76 It is probable that the mutation arose in a Norse or Celt who had a chromosome 6p that also carried HLA-A*03. Analyses of C282Y allele frequencies in different regions of Europe indicate that C282Y was spread by Viking exploration, much of which occurred during the interval of 793 to 1066. Other diaspora, including those of Europeans to North America, Australia, or New Zealand, promoted global spread of C282Y. The prevalence of HFE C282Y heterozygotes in western European and derivative white populations is 7% to 10%. This infers that heterozygosity for this allele was advantageous centuries ago. The slightly greater serum iron and ferritin measures in C282Y homozygotes observed in recent population screening studies34,35 suggest that C282Y was either an adaptation to a cereal diet or the effects of celiac disease in early Europeans77,78 or provided an evolutionary or natural selection advantage because it increased absorption of iron in women and its subsequent availability to their fetuses.79 Perhaps, the absorption of increased fractions of essential non-iron trace metals was a nutritional advantage of C282Y heterozygotes. HFE is an immune system gene, mutations of which may have increased resistance to plague, malaria, or other infections in early Europeans. Persuasive evidence that supports any of these hypotheses is lacking. Perhaps, very small differences between C282Y heterozygotes and those without C282Y that represented an advantage for heterozygotes over centuries are simply too small to detect today. It is also possible that HFE C282Y is a surrogate marker tightly linked to another chromosome 6p gene in which the true advantageous mutation lies.79

NON-HFE HEMOCHROMATOSIS The discovery of HFE in 1996 sparked renewed interest in the genetic and molecular mechanisms that control iron absorption and homeostasis and the clinical and molecular descriptions of uncommon types of hemochromatosis. Characteristics of HFE and non-HFE hemochromatosis syndromes are summarized in

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Table 4. Diagnosis and treatment of non-HFE hemochromatosis is similar to that of HFE hemochromatosis (Table 3).42 “Juvenile” hemochromatosis, sometimes called hemochromatosis type 2, is characterized by iron overload, often severe, that occurs in children or young adults. The most common subtype (sometimes called hemochromatosis type 2a) is an autosomal recessive disorder due to pathogenic mutations in the hemojuvelin gene (HJV or HFE2; chromosome 1q21.1) (Table 4). Each patient has 2 pathogenic HJV mutations (either homozygous or compound heterozygous configuration). Consanguinity has been identified in some affected families. The most common mutation is HJV G320V, a pathogenic allele that has been detected in juvenile-onset hemochromatosis kinships of diverse European ethnicities.88,89 About 30 pathogenic HJV mutations have been reported. Some HJV hemochromatosis patients also have an abnormal HFE genotype but the latter does not account for iron overload in most cases.90 Hemojuvelin is involved in the up-regulation of hepcidin synthesis, not in the regulation of HFE function. Decreased hemojuvelin activity decreases hepcidin synthesis, thus decreasing inactivation of ferroportin. Consequently, iron export from absorptive enterocytes into the plasma via ferroportin is increased, even in the presence of increased storage iron. HJV hemochromatosis is much more severe than hemochromatosis due to HFE C282Y homozygosity in subjects of the same or similar age. Males and females are usually affected equally. Symptoms include abdominal pain and hepatomegaly in children, hypogonadotrophic hypogonadism and arthropathy in teenagers and severe heart failure, cardiac arrhythmias and cirrhosis before the age of 30 years.91 Hepatic iron is deposited preferentially in hepatocytes. Iron deposition in the anterior pituitary gland, especially in gonadotroph cells and in cardiac myocytes and Purkinje fibers, accounts for hypogonadism and cardiomyopathy, respectively, in most cases. Based on the volume of blood removed to achieve iron depletion, it can be deduced that some patients absorbed 3.2 to 3.9 mg of dietary iron per day.92 In contrast, persons with severe HFE hemochromatosis usually absorb #2 mg of iron daily.93,94 Mutations in the hepcidin gene (HAMP; chromosome 19q13.12) cause a rare type of “juvenile” hemochromatosis (sometimes called hemochromatosis type 2b) (Table 4). Severity of iron overload and age of onset are variable. Hepatic iron

is deposited preferentially in hepatocytes. This disorder is transmitted as an autosomal recessive trait. The first reported HAMP mutation (nucleotide 208T/C) encodes substitution of a normal cysteine by arginine at amino acid 70 (C70R).80 At least 12 other deleterious HAMP mutations have been reported. Mutant hepcidin has decreased ability to bind to ferroportin, its only known receptor. Consequently, ferroportin is not degraded and thus continues to transport iron across the basolateral membrane of absorptive enterocytes in the presence of excess iron stores.95 Some patients with HFE mutations are heterozygous for pathogenic HAMP promoter or coding region mutations that contribute to the pathogenesis of iron overload (“digenic” hemochromatosis).38 Mutations of the transferrin receptor 2 gene (TFR2; chromosome 7q22.1) cause a rare autosomal recessive type of hemochromatosis (sometimes called hemochromatosis type 3) that occurs in persons of European or Asian ancestry (Table 4). Penetrance of TFR2 hemochromatosis is relatively great. The clinical phenotype is moderately variable and resembles that of either HFE hemochromatosis or “juvenile” hemochromatosis. Hepatic iron is deposited preferentially in hepatocytes. Approximately 20 pathogenic TFR2 mutations have been reported. Consanguinity has been identified in some affected families. TFR2 Y250X and R455Q have been detected in individuals or kindreds who were not closely related.83 Pathogenic TFR2 mutations result in either gain-of-function protein or decreased quantities of functional protein. Transferrin receptor 2 is thought to modulate the signaling pathway that controls hepcidin expression with consequent low hepcidin and increased iron absorption and deposition in hepatocytes. Ferroportin hemochromatosis (sometimes called hemochromatosis type 4) is an uncommon heterogeneous autosomal dominant disorder. It is caused by mutations of a gene of the solute carrier family 40 (member 1) (SLC40A1 or FPN1, chromosome 2q32.2), which encodes the synthesis of ferroportin, the only known receptor of hepcidin.84,85,96,97 There are 2 distinct ferroportin hemochromatosis phenotypes (Table 4). “Loss-of-function” SLC40A1 mutations (eg, A77D, V162del, G490D) encode ferroportin that either is not presented normally to the cell surface or has defective iron transport activity. Such mutations decrease iron absorption from the intestine and inhibit iron egress from macrophages.

TABLE 4. Characteristics of HFE and other hemochromatosis syndromesa Pathogenic Gene mutations (chromosome) Protein Mode of inheritance reported Phenotype penetrance; age HFE (6p21.3)

HFE

Autosomal recessive

;60

HAMP (19q13.12) Hepcidin

;13

HJV (1q21.1)

Autosomal recessive; “digenic” with HFE mutations Hemojuvelin Autosomal recessive

;30

Transferrin Autosomal recessive receptor 2

;20

TFR2 (7q22.1)

SCL40A1 (2q32.2) Ferroportin

a

Autosomal dominant

;40

Low; severe phenotypes rare before age 30 yr High; variable age of onset

Distribution

Reference

Common; western Europeans Rare; Europeans

17,34,47

High; usually early age of onset Rare; diverse European ethnicities High; variable age of onset Very rare; Europeans; Asians Variable, mild anemia with Uncommon; loss-of-function alleles; cosmopolitan variable age of onset

80

81,82

83

84,85

See detailed genetic and clinical information about these syndromes.86,87

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Phenotypes of affected patients include normal or low transferrin saturation, mild anemia and predominance of iron retention in macrophages, including Kupffer cells. “Gain-of-function” mutations (eg, Y64N, N114D, N144H, C326Y) encode ferroportin that cannot bind hepcidin normally or be internalized after hepcidin binding (hepcidin resistance). This increases iron absorption and stimulates iron export by macrophages. Affected patients have elevated transferrin saturation and preferential deposition of iron in hepatocytes.98 Ferroportin mutations are cosmopolitan. Most are restricted to single families. SLC40A1 V162del has been reported in persons with iron overload from Australia, Europe and Asia. SLC40A1 A77D has been reported in persons with iron overload in Italy, Australia and India. SLC40A1 Q248H occurs as a polymorphism in native peoples who reside in many regions of sub-Saharan Africa and in African Americans, but probably does not cause iron overload.99

SCREENING FOR HEMOCHROMATOSIS AND IRON OVERLOAD Population screening studies for hemochromatosis and iron overload have been performed and evaluated on a large scale in France, Australia, United States, Canada and Norway as government-funded research.28–30,32–35 These studies have generated voluminous data about many aspects of HFE hemochromatosis and other iron-related disorders. In the United States, further population screening is not recommended by public health personnel based in part on the tenet that knowledge about the natural history of hemochromatosis and optimal screening strategies is inadequate.100 The “pros” and “cons” central to population screening debates in the United States are summarized in Table 5. Population screening continues in some countries of Europe and in Australia.

TABLE 5. Population screening for HFE hemochromatosis and iron overload Pros Cons Hemochromatosis-associated genotype(s) common Definitive phenotypic, genotypic tests available Early diagnosis, treatment prevent known complications Treatment effective, inexpensive, widely available High burden of disease in some patients, especially those with liver complications

;90% of C282Y homozygotes never develop iron overload disease Which test(s) has greatest sensitivity? is most specific? is least expensive? Few homozygotes develop progressive iron overload Overall cost great; relatively few may benefit Diagnosis, treatment may be late: 5-fold increase in risk of iron overload death if serum ferritin .1000 mg/L, even with treatment Cost per case diagnosed too high

Rare iron overload disorders would be detected Phlebotomy blood may be Ethical, practical problems with suitable for transfusion, would therapeutic “blood donation” augment blood supply White males at greatest risk for Little benefit for women, noniron overload disease white subpopulations Iron deficiency, non-iron These conditions should be hyperferritinemia are diagnosed in practice settings common, would be detected

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Benefits of “screening” for hemochromatosis in specific clinics or practices (eg, hepatology, diabetology), if any, remain unproven. “Screening” of first-degree family members of hemochromatosis probands, on the other hand, has a high probability of identifying other persons with hemochromatosis or iron overload, some of whom would benefit from further evaluation and treatment.101 Iron phenotyping and HFE mutation analyses yield different but complementary results.101

UNANSWERED QUESTIONS

Environmental/acquired and heritable “modifiers” would explain the phenotypic heterogeneity characteristic of HFE C282Y homozygotes, but many putative “modifiers” remain undiscovered. Many environmental or acquired modifiers of iron absorption and metabolism are well understood (eg, dietary iron content and non-iron composition, growth and development, blood loss) but are often difficult to ascertain and quantify for analysis. Recent studies that sought to identify non-HFE iron-related “modifier” mutations through sequencing or allele-specific analyses of non-HFE iron genes have detected some mutations of interest, but most of them are too rare to account for effects on HFE hemochromatosis phenotypes. Current approaches to identifying putative modifiers include exome or whole-genome sequencing. Iron overload severity explains a minority of the risk of cirrhosis in persons with HFE C282Y homozygosity. Thus, susceptibility of hemochromatosis patients to cirrhosis is heterogeneous. Acquired liver conditions such as alcoholic liver disease, non-alcoholic fatty liver and chronic viral hepatitis C act in synergy with hepatocellular iron overload caused by hemochromatosis to induce cirrhosis. Nonetheless, these and other known liver conditions, along with hepatic iron overload, are not significantly associated with cirrhosis risk in most hemochromatosis cohorts. It is plausible but unproven that mutations in genes that encode proteins that regulate iron deposition in hepatocytes or stellate cells, protect liver from freeradical injury or regulate proliferation or synthetic activities of hepatic stellate cells influence cirrhosis risk. In the clinical realm, it would be valuable to learn in controlled trials whether limited “screening” for HFE hemochromatosis is beneficial for high-risk population subgroups, especially in white men aged 30 to 50 years. Increasing evidence suggests that there are positive associations of iron overload severity and risk of hemochromatosis arthropathy. This infers that phlebotomy therapy presently not advocated for some patients with hemochromatosis, if performed, would decrease the frequency or severity of future arthropathy. Mini-hepcidins and small-interfering RNAs (eg, siRNA antiTMPRSS6) are being developed in mouse models as possible therapeutic agents for managing iron overload in hemochromatosis and beta-thalassemia humans, but clinical studies of safety, efficacy and cost have not been performed. Finally, we must continue to educate medical students, physicians and the public about iron, hemochromatosis and hepcidin, although devising optimal educational strategies and assessing their results will be challenging. ACKNOWLEDGMENTS The author expresses his sincerest appreciation to Dr. Robert T. Means, President of Southern Society of Clinical Investigation, 2012 to 2103, whose kind invitation to participate in the Presidential Symposium inspired this work. Ms. Joan Kemp, Executive Director of Southern Society of Clinical Investigation, provided valuable assistance for Symposium

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preparation. Dr. Cindy N. Roy critically reviewed this work and made constructive suggestions. The author also recognizes the stimulation, encouragement and collaboration of these persons (in alphabetic order): Ronald T. Acton, Paul C. Adams, Bruce R. Bacon, J. Clayborn Barton, Luigi F. Bertoli, Ernest Beutler, Robert S. Britton, Eugénia Cruz, Corwin Q. Edwards, The HEIRS Study, Pauline L. Lee, Pradyumna D. Phatak, Graça Porto, Maria de Sousa and Ketil Thorstensen. REFERENCES 1. Stahl GE. Experimenta, observationes, animadversiones. Chymicae et Physicae (Berlin) 1731;CCC Numero:281–3. 2. Virchow R. Die patholgischen pigmente. Arch Pathol Anat 1847;1: 379–486. 3. Perls M. Nachweis von eisenoxyd in gewissen pigmenten. Virchow Arch Pathol Anat 1867;39:42–8.

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