Hemochromatosis Thomas W. Faust, M.D., M.B.E. Professor of Clinical Medicine Division of Gastroenterology The University of Pennsylvania

Hemochromatosis Introduction Classification of iron overload syndromes Pathogenesis Clinical manifestations Diagnosis Treatment Screening

Hereditary Hemochromatosis Introduction Inherited disorder of inappropriate dietary iron absorption Prevalent in 1/250 individuals Most patients are asymptomatic Hepatic and extrahepatic manifestations HFE (autosomal recessive) mutations account for majority of cases Non-HFE mutations are rare causes of iron overload Secondary iron overload states C282Y homozygotes account for 80-95% of cases associated with HFE mutations. H63D and S65C account for other HFE mutations. Iron overloading typically with C282Y. Only 70% of C282Y homozygotes will develop iron overload. Modifying genes may account for lack of complete penetrance. Consider repeating TS and ferritin every 5 yrs for non-expressing C282Y homozygotes. C282Y/H63D or H63D/H63D usually have mild to moderate iron overload; most pts with these mutations with severe iron overload have another contributing disease (ETOH, viral hepatitis etc). Likewise, C282Y and H63D heterozygotes usually have normal iron stores; elevated iron associated with another unidentified mutation or another disease. Modifier genes may account for incomplete penetrance. Disease is more common in males. Non-HFE causes of iron overload: hepcidin, hemojuvelin, TR2, ferroportin. Other causes of iron overload: ineffective erythropoiesis, chronic liver disease, parenteral iron administration. Phatak P et al., Ann Intern Med 2008;149:270-272

Hemochromatosis Hereditary Causes HFE hemochromatosis C282Y/C282Y (95%) C282Y/H63D (4%) H63D/H63D or C282Y/S65C (1%) Non HFE hemochromatosis (rare) Hemojuvelin Hepcidin Ferroportin Transferrin receptor 2 DMT-1 Juvenile HH: mutations of hemojuvelin and HAMP. Pts with hemojuvelin mutations have low hepcidin. Inactivating mutations of HAMP that encodes for hepcidin. Inactivating mutations for ferroportin result in iron retained in RE cells rather than hepatocytes; transferrin saturation lower, hepcidin increased. Gain of function mutations of ferroportin result in classic HH phenotype; hepcidin increased. Ferroportin disease: Autosomal dominant. Accumulation of iron in macrophages. Elevated ferritin with mild hypochromic microcytic anemia. TS may be normal or elevated. Liver disease less advanced when compared to HFE dz.1 unit phlebotomy monthly Juvenile hemochromatosis:Autosomal recessive. Mutations in HJV or HAMP (hepcidin) genes. Severe iron overload in heart, endocrine organs, and liver. Treatment is phlebotomy Beutler E. et al, Lancet 2002;359:211-218

Hemochromatosis Non-Hereditary Causes Secondary iron overload Thalassemia major Sideroblastic anemias Liver disease (ETOH, HCV, HBV, PCT, NAFLD) Excessive iron ingestion Parenteral iron overload RBC transfusions Iron-dextran infusions Long-term dialysis ETOH, HBV, HCV, and NAFLD can have elevation in ferritin and/or TS without iron overload. Bx justified for clarification.

Duodenal Iron Absorption Crypt cells HFE-transferrin receptor complex senses body iron stores Upregulation or downregulation of DMT-1 based upon body iron stores Villous cells Dietary iron absorption occurs via DMT-1 and ferroportin Transporter expression based upon body iron stores sensed by crypt cells HFE-TFR-1 complex senses iron stores in crypts. Bacon B et al, Gastroenterology. 1999;116:193-207.

Regulation of Iron Absorption Crypt Cell Model HFE-transferrin receptor acts as sensor of iron stores in Crypt DMT-1 synthesized based upon iron stores Iron absorbed at villus tip Crypt cell model — The findings noted above have led to the postulate that the role of the HFE protein is to modulate the uptake of transferrin-bound iron into crypt cells, allowing the complex to act as a sensor of body iron stores [59-62]. (See "Regulation of iron balance".) In this proposed model of the regulation of iron absorption, the following steps would occur (figure 2). Increased body iron stores and the accompanying rise in transferrin-bound iron lead to enhanced uptake of iron into crypt cells. As a result of the ensuing increase in intracellular iron, the differentiating enterocytes migrating up to the villus tip downregulate the production of the iron transporter DMT1, reducing the absorption of dietary iron. This process is reversed in iron deficiency as iron transporters in the developing villus cells and iron absorption are increased. In hereditary hemochromatosis, mutations in HFE may impair TfR-mediated uptake of transferrin-bound iron into crypt cells, providing a false signal that iron stores are low. Ferric iron converted to ferrous form. Ferrous iron transported by DMT-1 across apical membrane. Iron in enterocyte stored as ferritin and sloughed with enterocyte or transferred to plasma via ferroportin. Basolateral transfer requires oxidation to ferric form via hephaestin. Zoller H. et al, Lancet. 1999;353:2120-2123.

Regulation of Iron Stores Influence of Hepcidin Regulation of ferroportin-mediated iron export from enterocyte Regulation of ferroportin-mediated iron export from macrophages Hepcidin is a hepatic peptide that may be the key regulator of iron metabolism. Hepcidin deficiency in knockout mice results in severe iron overload.[10] Hepcidin may also have a direct effect on the uptake of iron by intestinal epithelia.[11] Conversely, a relative deficiency of hepcidin can result in increased iron efflux from macrophages. After hepcidin binds ferroportin, ferroportin is internalized and degraded. Fig. 64-2  Schematic representation of the HFE protein, a class 1 MHC protein that is involved in iron metabolism with a cascade of other iron proteins. Typical hemochromatosis patients are homozygous for the C282Y mutation of HFE, which cause conformational changes in the protein that impair intracellular trafficking. BMP, bone morphogenetic protein; Fe, iron; HJV, juvenile hemochromatosis gene Hepcidin is a hepatic peptide that may be the key regulator of iron metabolism. Hepcidin deficiency in knockout mice results in severe iron overload.[10] Hepcidin may also have a direct effect on the uptake of iron by intestinal epithelia.[11] Conversely, a relative deficiency of hepcidin can result in increased iron efflux from macrophages. This is consistent with the paradoxical observation in HFE-related hemochromatosis of relative iron deficiency in the spleen and within macrophages. Hepcidin expression in liver tissue, serum, and urine has suggested a relative deficiency in HFE-linked hemochromatosis.[12] However, at the present time it is unclear whether the defect in hepcidin is the primary abnormality in hemochromatosis, or a downstream effect of abnormalities in the HFE protein. The control of hepcidin may be related to a cellular iron-sensing mechanism that includes bone morphogenetic protein-6 (BMP6), transferrin receptors 1 and 2, HFE protein, hemojuvelin, and Smad proteins (Fig. 64-2).[13-16] It is intriguing to speculate whether alterations in non-HFE iron proteins could explain the wide range of clinical expression seen in hemochromatosis. The concept is that the most severe cases of HFE-related hemochromatosis may be heterozygotes for a mutation in another iron-related protein. These mutations would need to be very common to fit this hypothesis. A tantalizing theory has been suggested that the hemochromatosis gene has not been found yet and that the HFE gene is only a modifying gene.[17] Ganz T. Cell Metab 2008;7:288-290

Regulation of Iron Stores Normal Hepcidin regulates ferroportin-mediated iron export from duodenum, macrophages, and liver BMP, HJV, HFE, and TFR2 sense body iron stores and regulates release of hepcidin Loss of appropriate hepcidin expression is the central pathogenic mechanism in hereditary hemochromatosis. (A) In normal humans, iron is sensed by the liver and regulates hepcidin secretion, which in turn interacts with and downregulates ferroportin (FPN) expressed on the surfaces of macrophages and duodenal enterocytes, and hepatocytes to regulate the release of iron into the blood stream. Hepatic bone morphogenetic protein (BMP, BMP6 in mice), hemojuvelin (HJV), HFE, and transferrin receptor 2 (TFR2) are all important for sensing serum iron levels and adjusting the expression of hepcidin appropriately to maintain iron balance. (B) Inherited mutations in any one of these hepcidin regulators leads to the inability of the liver to properly sense iron levels, and consequently to inappropriately low expression of hepcidin, unregulated ferroportin activity, and excess export of iron into the bloodstream. The excess iron can deposit into parenchymal tissues where it causes end organ damage that is characteristic of hemochromatosis. Nemeth E. et al, Science 2004;306:2090–2093

Hemochromatosis Non-Ferroportin Mutations Mutations of BMP, HJV, HFE, and TFR2 alter ability of liver to sense body iron stores Inappropriately low level of hepcidin Excess ferroportin-mediated export of iron from duodenum, macrophages, and liver Loss of appropriate hepcidin expression is the central pathogenic mechanism in hereditary hemochromatosis. (A) In normal humans, iron is sensed by the liver and regulates hepcidin secretion, which in turn interacts with and downregulates ferroportin (FPN) expressed on the surfaces of macrophages and duodenal enterocytes, and hepatocytes to regulate the release of iron into the blood stream. Hepatic bone morphogenetic protein (BMP, BMP6 in mice), hemojuvelin (HJV), HFE, and transferrin receptor 2 (TFR2) are all important for sensing serum iron levels and adjusting the expression of hepcidin appropriately to maintain iron balance. (B) Inherited mutations in any one of these hepcidin regulators leads to the inability of the liver to properly sense iron levels, and consequently to inappropriately low expression of hepcidin, unregulated ferroportin activity, and excess export of iron into the bloodstream. The excess iron can deposit into parenchymal tissues where it causes end organ damage that is characteristic of hemochromatosis. Nemeth E. et al, Science 2004;306:2090–2093

Hemochromatosis Ferroportin Mutations Loss of function Limited ability to export iron Accumulation of iron in macrophages Hepcidin increased Gain of function Resistant to inhibitory effects of hepcidin Phenotypically similar to classic hemochromatosis Some mutations produce a molecule which either does not traffic appropriately to the cell surface or which has limited ability to export iron, as in panel D of the figure (figure 3). These "loss of function" mutations result in excess accumulation of iron in macrophages, with resulting high serum ferritin, normal to reduced transferrin iron saturation, and a mild anemia. Other mutations produce a ferroportin resistant to inhibition by the iron regulatory molecule hepcidin, retaining full iron export capability, as in panel E of the figure (figure 3) [111-113]. These "gain of function" mutations allow iron to be absorbed in excess of need, with patients manifesting high levels of ferritin and hepcidin, increased transferrin saturations, and typical deposition of iron in the hepatic parenchyma. Letocart E. et al, Br J Haematol. 2009;147(3):379

Hemochromatosis Overview of Clinical Manifestations Asymptomatic state (majority) Abnormal iron studies and liver function tests Non-specific systemic symptoms Weakness, fatigue, lethargy, apathy, weight loss, abdominal pain Organ-related disease Hepatic manifestations Extrahepatic manifestations

Hemochromatosis Hepatic Manifestations Hepatosplenomegaly Micronodular cirrhosis Portal hypertensive bleeding Ascites/SBP/HRS Encephalopathy Hepatocellular carcinoma www.gfmer.ch/genetic_diseases_v2/gendis_detail_list.php?cat3=821

Hemochromatosis Physical Examination Physical findings in patients with progressive liver disease Findings not specific to hemochromatosis

Hemochromatosis Hepatocellular Carcinoma Patients with cirrhosis at risk for HCC. All patients with cirrhosis should be screened per AASLD guidelines OLT considered for cirrhotic patients with HCC Netter’s Gastroenterology, 2nd ed., Elsevier Inc., 2010, all rights reserved

Hemochromatosis Extrahepatic Manifestations Cardiac Restrictive/dilated cardiomyopathy Arrhythmias Rheumatologic Arthalgias/arthritis Chondrocalcinosis Osteoporosis Dermatologic Hyperpigmentation Porphyria cutanea tarda Endocrinologic Diabetes Loss of libido/impotence Amenorrhea Hypothyroidism Infectious Yersinia, pasteurella, vibrio vulnificus, listeria Joint involvement: PIP joints, shoulders, wrists, knees, feet. Hyperpigmentation usually melanin, not iron. Endocrine abnormalities may be associated with low FSH, LH, and testosterone. Iron deposited in parenchymal cells of liver, pancreas, heart, endocrine glands, skin, joints.

Hemochromatosis Systemic Disorder Multiple organs involved with progressive iron overload Therapeutic phlebotomy may correct some of the clinical manifestations Netter’s Gastroenterology, 2nd ed., Elsevier Inc., 2010, all rights reserved

Hemochromatosis Extrahepatic Manifestations

Diagnosis of Hemochromatosis Iron Studies Transferrin Saturation Ferritin Value ≥ 45% most common early phenotypic marker Sensitivity > 90% Fasting value preferable F/U with genetic testing Rises with progressive iron overload F/U with genetic testing Other diseases ETOH, NAFLD, viral hepatitis, inflammatory disorders, neoplasms Predicts fibrosis Normal ferritin in combination with TS < 45% has NPV of 97%. Iron overload may be present in patients with elevated ferritin and normal TS particularly in non-HFE or C282y/H63D iron overload states. Ferritin > 1000 in combination with AST/ALT elevation predicts cirrhosis in 80% of C282Y homozygotes. Ferritin < 1000 in combination with normal AST/ALT an accurate predictor for absence of fibrosis/cirrhosis. If either TS > 45% or ferritin above ULN, get HFE mutation analysis. Beutler E. et al, Lancet 2002;359:211-218

Diagnosis of Hemochromatosis Genetic Testing C282Y/C282Y For all patients with TS ≥ 45% For all patients with elevated ferritin Liver biopsy for ferritin ≥ 1000 μg/L or elevated AST/ALT C282Y/H63D or other For all patients with TS ≥ 45% For all patients with elevated ferritin Exclude other liver or hematologic diseases Testing for non-HFE mutations not widely available Consider liver biopsy Other constitutes C282Y/wt, H63D/wt, H63D/H63D, wt/wt. No difference in prevalence of C282Yor H63D in other liver diseases associated with elevated iron indices (viral hepatitis, NAFLD, PCT etc). Guyader D. et al, Gastroenterology 1998;115:929-936

Liver Biopsy Diagnosis of Hemochromatosis or Fibrosis Not required C282Y/C282Y Ferritin < 1000 μg/L Normal AST/ALT Required or suggested C282Y/C282Y, ferritin ≥ 1000 μg/L, elevated AST/ALT Consider for C282Y/H63D or other Routine histopathology Qualitative and quantitative iron assessment Other constitutes C282Y/wt, H63D/wt, H63D/H63D, wt/wt. HII not frequently used as it does not adequately discriminate HH from other causes of iron overload. Value of 1.9 for HII used. HII replaced by genetic testing. Adams P et al, J Lab Clin Med 1997;130:509-514

Hemochromatosis Liver Histopathology Hepatocytes Progressive iron accumulation from periportal (zone 1) to pericentral (zone 3) regions Routine H&E and Prussian blue stains Kupffer and biliary epithelial cells Iron accumulation with progressive iron loading Fibrosis and cirrhosis Trichrome stain Associated with advanced iron overload Hepatocellular carcinoma Iron-dependent oxidative damage and lipid peroxidation of hepatocytes with generation of profibrogenic cytokines from Kuppffer cells. Cytokines stimulate stellate cells to lay down collagen.

Hepatic Iron Overload Other Diseases ETOH, NAFLD, viral hepatitis, PCT, parenteral Panlobular and patchy iron distribution Iron accumulation in hepatocytes and Kupffer cells Iron accumulation is usually mild Ferroportin disease associated with iron overload in macrophages and reticuloendothelial cells

Hemochromatosis Pathology Progressive hepatic iron overload Myocardial iron overload Cirrhosis: H&E Cirrhosis: Prussian Blue www.gfmer.ch/genetic_diseases_v2/gendis_detail_list.php?cat3=821

Hemochromatosis Imaging CT MR Taouli B et al, AJR 2009;193:14-27 Jensen P, Br J Haematol 2004 Mar;124(6):697-711

Hemochromatosis Treatment Weekly or twice weekly phlebotomy Removal of 2-3 units per week or 0.5 units every other week Check Hgb/HCT prior to each phlebotomy Follow TS and ferritin every 3 mo. Endpoints: TS < 50%, ferritin < 50 μg/L Maintenance phlebotomy: 1 unit every 3 mo. Avoid iron deficiency Imaging and AFP every 6 mo. to screen for HCC in cirrhotic patients OLT for hepatic decompensation or HCC 500 cc of whole blood equals 250 mg of iron. Phlebotomy prior to cirrhosis and DM prolongs survival. Survival post OLT comparable to non-HH indications. Chelating agents in combination with phlebotomy may be beneficial for HH with cardiac iron overload. Bacon B. Gastroenterology 2001;120:718-725

Hemochromatosis Response to Phlebotomy Improvement Tissue iron stores Survival in absence of cirrhosis and DM Liver-associated enzymes Cardiac function, DM, skin pigmentation, fibrosis No improvement Established cirrhosis Arthropathy Testicular atrophy

Hemochromatosis Screening Family Screening Population Screening HFE (C282Y, H63D) and iron testing for first degree relatives C282Y/C282Y and C282Y/H63Y relatives with iron overload undergo phlebotomy C282Y/wt, H63D/H63D, H63D/wt not at risk for iron overload C282Y/C282Y or C282Y/H63D children undergo yearly ferritin assessment Role for population screening with genetic testing unclear Incomplete penetrance raises questions about clinical utility, cost effectiveness, and genetic discrimination Non HFE hemochromatosis is rare and genetic testing only available in research labs If iron studies normal, yearly f/u with iron studies warranted in patients with appropriate genetic mutations. Phenotypic expression can vary among patients with genetic mutations. For pts with iron overload and non-HFE hemochromatosis or secondary iron overload, phlebotomy warranted as for typical HH pt. Tavil A. Hepatology 2001;33:1321-1328

Hemochromatosis Algorithm Symptomatic Transferrin saturation/ferritin TS < 45% and normal ferritin No further evaluation TS ≥ 45% and/or elevated ferritin HFE genotype Asymptomatic HFE Genotype Adult 1st degree relative of HH Hemochromatosis Algorithm Bacon B et al, Hepatology,2011;54:328-343

Hemochromatosis Algorithm HFE genotype C282Y/H63D, C282Y heterozygote, non-C282Y Exclude other liver or hematologic diseases. ± liver biopsy Therapeutic phlebotomy C282Y/C282Y Ferritin < 1000 μg/L and normal liver enzymes Ferritin ≥ 1000 μg/L or elevated liver enzymes Liver biopsy for HIC and histopathology ± + Bacon B et al, Hepatology,2011;54:328-343

Hemochromatosis Take Home Points HFE (C282Y/C282Y) mutations account for most cases of hereditary hemochromatosis. Be aware of other non-HFE inherited and secondary causes of iron overload. Interrelationship between duodenal iron absorption and hepcidin is important. Patients can present with a variety of hepatic and extrahepatic manifestations. Diagnosis based upon iron studies, genetic testing, and liver biopsy. Phlebotomy is mainstay of therapy Genetic testing recommended for family members of patients with hereditary hemochromatosis.

Hemochromatosis Question 1 A 55 yr old man presents with mildly elevated transaminases. His serum ferritin is 3000 mcg/L, with transferrin saturation exceeding 90%. He is homozygous for C282Y. Liver ultrasound is normal and liver biopsy shows bridging fibrosis and markedly elevated hepatocellular iron. Weekly phlebotomy is started. The patient’s wife is heterozygous for C282Y and one normal allele. The patient has 2 sons ages 26 and 18. The older son’s ferritin is 2500 mcg/L, whereas the younger son’s ferritin is 180 mcg/L. At this time you make all of the following recommendations except: DDSEP 6, AGA Press, 2011.

Hemochromatosis Question 1 A. The older son should be tested for C282Y mutation of HFE gene B. For the older son, liver biopsy may be justified to R/O cirrhosis C. The younger son should be tested for C282Y mutation D. For the younger son, liver biopsy may be justified to R/O cirrhosis E. The older son should have liver ultrasound DDSEP 6, AGA Press, 2011.

Hemochromatosis Question 2 A baby born to parents of Irish descent (father homozygous for C282Y, mother genetic status unknown) is found to be homozygous for C282Y. What is the lifetime risk of hepatic decompensation and/or hepatocellular carcinoma if the child is carefully followed and undergoes phlebotomy over his/her lifetime? A. Zero B. 5% C. 20% D. 50% E. 80% DDSEP 6, AGA Press, 2011.

Hemochromatosis Question 3 An autosomal dominant form of hemochromatosis accompanied by high levels of hepcidin production is associated with mutations of the gene coding for which of the following? A. HFE protein B. Transferrin receptor type 2 C. Hemojuvelin D. Hepcidin E. Ferroportin DDSEP 6, AGA Press, 2011.

Hemochromatosis Question 4 Which one of the following statements about liver biopsy in patients with HFE (C282Y homozygous) hemochromatosis is correct? A. Liver biopsy should be performed in all patients after genetic testing. B. Kupffer cells take up iron before hepatocytes in patients with HFE (C282Y homozygous) hemochromatosis. C. Panlobular patchy iron accumulation in hepatocytes and Kupffer cells is classic for C282Y homozygous disease. D. In patients with C282Y disease, iron is taken up by periportal (zone 1) hepatocytes prior to pericentral (zone 3) hepatocytes. E. Bridging fibrosis is seen in early disease

Hemochromatosis Question 5 A 65 yr old male is recently diagnosed with C282Y hemochromatosis and cirrhosis. His transferrin saturation is 95% and his ferritin is 5284 mcg/L. Which one of the following statements is incorrect? A. He will not require surveillance for hepatocellular carcinoma once iron stores have been removed from the liver by phlebotomy. B. Phlebotomy should be initiated to achieve endpoints of T. Sat of < 50% and ferritin < 50 μg/L. C. Phlebotomy may improve cardiac function and glucose intolerance. D. Arthropathy does not usually improve with phlebotomy E. At the beginning of treatment, weekly or twice weekly phlebotomy is necessary to reach desired endpoints.

Hemochromatosis Question 6 Which one of the following statements about HFE hemochromatosis is correct? A. C282Y/H63D disease is the most common genetic abnormality. B. H63D/H63D commonly results in iron overload. C. Population-based screening for genetic hemochromatosis is recommended. D. CT and MR of the liver are sensitive tests for diagnosing disease. E. Hepcidin regulates ferroportin-mediated iron export from duodenal enterocytes.

Hemochromatosis Answers to Questions 1. D 2. A 3. E 4. D 5. A 6. E