Genes and Metabolic Liver Disease: Hemochromatosis Bruce R. Bacon, M.D. James F. King M.D. Endowed Chair in Gastroenterology Professor of Internal Medicine Division of Gastroenterology and Hepatology Saint Louis University Liver Center St. Louis, Missouri

History of Hemochromatosis 1865 – Trosier – first case 1889 – von Recklinghausen – hemochromatosis 1935 – Sheldon – inherited defect in iron metabolism 1976 – Simon – HLA-A3, short arm chromosome 6 1996 – Mecator Genetics – HFE 1997 – present DMT-1 Ferroportin Transferrin receptor-2 Hemojuvelin Hepcidin

Classification of Inherited Iron Overload Syndromes Hereditary Hemochromatosis HFE-related C282Y/C282Y C282Y/H63D Other HFE mutations Non-HFE-related Hemojuvelin (HJV) Transferrin receptor-2 (TfR-2) Ferroportin (SLC40A1) Hepcidin (HAMP) African iron overload

Classification of Iron Overload Syndromes - 2 Secondary Iron Overload Iron-loading anemias Thalassemia major Sideroblastic Chronic hemolytic anemia Aplastic anemia Pyruvate kinase deficiency Pyridoxine-responsive anemia Parenteral iron overload Red blood cell transfusions Iron-dextran injections Long-term hemodialysis Chronic liver disease Porphyria cutanea tarda Hepatitis C Hepatitis B Alcoholic liver disease Nonalcoholic steatohepatitis Following portocaval shunt Dysmetabolic iron overload syndrome Miscellaneous Neonatal iron overload Aceruloplasminemia Congenital atransferrinemia

HFE Genotype in Patients with Hemochromatosis Study Feder, et al. Beutler, et al Jouanolle, Jazwinksa, Carella, Adams and Chakrabarti Bacon, Country USA France Australia Italy Canada No. of Patients 178 147 65 112* 75 128 66 Genotype n(%) C282Y 148 (83) 121 (82) 59 (91) 112 (100) 48 (64) 122 (95) 60 (91) C282Y** H63D 8 (4) 8 (5) 3 (5) 2 (2.27) 2 (1.6) 1 (1.5) Wild Type 1 (0.5) 2 (1) 1 (1.3) Wild type 7 (4) 4 (3) 2 (3) 3 (4) 13 (7) 10 (7) 16 (21) 4 (3.1) *All patients had a family history of iron overload ** Compound heterozygote

Ser65  Cys, S65C His63  Asp, H63D  Heavy Chain 1 2 Cys282  Tyr, C282Y  Heavy Chain Ser65  Cys, S65C 1 2 NH2 2-microglobulin Extracellular HOOC Plasma membrane Cytosol 3 Nature Genetics 13: 309-408, 1996

HFE Genotype in Patients with Hemochromatosis 771 patients with phenotypic HH 670 (86.9%) with C282Y/C282Y 44 (5.7%) with C282Y/H63D 24 (3.1%) with C282Y/wt

Incidence of Hereditary Hemochromatosis 1 in 200 - 250 individuals of Northern European descent 11% heterozygotes Estimated 600,000 to 1,000,000 afflicted Americans Estimated 27 million heterozygotes

Hereditary Hemochromatosis - Diagnosis Requirements of Diagnosis Suspicion, serum iron studies Liver biopsy Use of HII Differential diagnosis Alcoholic liver disease Chronic viral hepatitis Nonalcoholic steatohepatitis Genetic test

Typical Symptoms in Patients with HH % Weakness, lethargy, fatigue 40-85 Apathy, lack of interest Abdominal pain 30-60 Weight loss Arthralgias 40-60 Loss of libido, impotence Amenorrhea 20-60 Congestive heart failure symptoms 0-40

Common Physical Findings in HH % Hepatomegaly 60-85 Cirrhosis 50-95 Skin pigmentation 40-80 Arthritis (second, third metacarpophalaneal joints) 40-60 Clinical diabetes 10-60 Splenomegaly 10-40 Loss of body hair 10-30 Testicular atrophy Dilated cardiomyopathy 0-30

Hereditary Hemochromatosis Symptoms and Physical Findings (%) No symptoms 73 Lethargy, and/or weakness 25 Loss of libido, impotence 12 Arthralgias 13 Diabetes 5 Skin pigmentation Am J Gastroenterol 92:784-789, 1997

Principal Clinical Features in Hereditary Hemochromatosis Milder et al. 1980 Edwards Niederau 1985 Adams 1991 Bacon & Sadiq 1997 Number of subjects 34† 35* 163* 37‡ 40 Symptoms (#) Weakness, lethargy 73 20 83 19 25 Abdominal pain 50 23 58 3 Arthralgias 47 57 43 13 Loss of libido, impotence 56 29 38 32 12 Cardiac failure symptoms 35 15 Physical and Diagnostic Findings (%) Cirrhosis (biopsy) 94 69 Hepatomegaly 76 54 Splenomegaly - Loss of body hair 6 Gynecomastia 8 Testicular atrophy 14 Skin pigmentation 82 75 9 5 Clinical diabetes 53 55 11 * Patient selection occurred by both clinical features and family screening. † Only symptomatic index cases were studied. ‡ Discovered by family studies. Zakim Boyer, 1996;1453.

Evaluation of Iron Stores Serum iron, TF saturation and ferritin Liver biopsy for stainable iron, biochemical determination of iron Noninvasive imaging modalities Computed tomography Magnetic resonance imaging Magnetic susceptibility Iron removed by phlebotomy (1 U=250 mg)

Blood Iron Studies in HH Normal HH Serum iron (µg/dl) 50-150 180-300 Transferrin (mg/dl) 250-370 200-300 Transferrin saturation (%) 20-50 80-100 Serum ferritin (ng/ml) Males 20-300 500-6,000 Females 15-250

Hemochromatosis – Role of Liver Biopsy In the past – establish diagnosis Determine degree of fibrosis, cirrhosis Determine other abnormalities Phenotypic variability Recommended if: Ferritin > 1000 ng/mL ALT, AST elevated Hepatomegaly Age > 40 years

Therapeutic Phlebotomy for HH Clearly improves survival Prepare patients for up to 6 – 12 months Iron burden in men greater than in women despite initial HIC Difficult to predict phlebotomy requirements Each unit of blood – approximately 30 ng/mL ferritin

Therapeutic Phlebotomy for HH Weekly phlebotomy Hct. > 35% before each one Ferritin to 50 to 100 ng/mL Transferrin saturation to < 50%

Maintenance Phlebotomy for HH Most patients – one unit Q 2-3 months TS < 50%; ferritin < 100 ng/mL One unit of whole blood = 250 mg iron Most patients absorb 2 to 3 mg/day, more than needed Some patients – no re-accumulation

Hereditary Hemochromatosis Response to Therapy Edwards et al. Ann Int Med 93:519-525, 1980

Results of Therapy Reduction to normal tissue iron stores. Improved survival if diagnosis and treatment before development of cirrhosis and diabetes. Improved sense of well-being, energy level. Improved cardiac function. Improved control of diabetes. Reduction in abdominal pain. Reduction in skin pigmentation. Normalization of elevated liver enzymes. Reversal of hepatic fibrosis (approximately 30% of cases). No reversal of established cirrhosis. Reduction in portal hypertension in cirrhotics. No (or minimal) improvement in arthropathy. No reversal of testicular atrophy.

Hereditary Hemochromatosis: Survival N Engl J Med 313:1256-1262, 1985

Hereditary Hemochromatosis: Survival with Cirrhosis N Engl J Med 313:1256-1262, 1985

HH – Family Screening HFE mutation analysis has replaced HLA-typing Practically – HFE mutation analysis, TS, and ferritin all at once

HH – Family Screening For analysis of risk in children – perform mutation analysis in spouse (or other parent) first May be able to avoid testing in children Adams, Clin Genet 53:176-178, 1998

General Population Studies in HH Transferrin saturation, ferritin, CBC HFE mutation analysis Several studies from around the world

Population Screening for Hemochromatosis 41,038 adults screened in San Diego Health appraisal unit CBC, transferrin saturation, ferritin level, HFE genotype Questionnaire Beutler et al., Lancet 359:211-218, 2002

Population Screening for Hemochromatosis 152 C282Y/C282Y 616 C282Y/H63D 67% of C282Y/C282Y had elevated ferritin No difference in symptoms from controls 1 of 152 with signs and symptoms of hemochromatosis Beutler et al., Lancet 359:211-218, 2002

Population Screening for Hemochromatosis Beutler et al., Lancet 359:211-218, 2002

Prevalence of homozygotes C282Y homozygotes with a normal ferritin (%) Prevalence of C282Y Homozygotes Without Iron Overload in Screening Studies Population sample Country n Prevalence of homozygotes C282Y homozygotes with a normal ferritin (%) Electoral roll New Zealand 1,064 1 in 213 40 Primary care USA 1,653 1 in 276 50 Epidemiological survey Australia 3,011 1 in 188 25 Blood donors Canada 4,211 1 in 327 81 General public 41,038 1 in 270 33 North America 44,082 1 in 227 29,676 1 in 146 32 Total 124,636 1 in 240 41

Hereditary Hemochromatosis Patients with C282Y/C282Y mutation without iron overload Environmental factors Nutritional deficiency, malabsorption Gastrointestinal blood loss Menstrual blood loss Pregnancy Genetic factors Hepcidin DMT-1 Ferroportin TfR-2 Hemojuvelin Others

Hereditary Hemochromatosis Patients with iron overload 10% to 15% are negative for C282Y/C282Y Mutations in other genes Ferroportin TfR-2 Hepcidin Hemojuvelin Others

Iron Overload Syndromes Ferroportin (SLC40A1) 2q32 Autosomal dominant RE cell distribution African iron overload, Solomon Islanders Pedigree described Pietrangelo et al., NEJM 341:725-732, 1999 Montosi et al., JCI 108:619-623, 2001 Gordeuk et al., Blood Cells Mol Dis 31:299-304, 2003

Iron Overload Syndromes Transferrin receptor-2 (TfR-2) 7q22 Autosomal recessive Parenchymal cell distribution Pedigree described Hoffman et al., Blood 100:1099-1100, 2002

Iron Overload Syndromes Hepcidin (HAMP) 19q13.1 25 amino-acid peptide Juvenile iron overload (rarely) Autosomal recessive Parenchymal cell distribution More importantly – principal “hormone” involved in iron regulation Roetto et al., Nat Genet 33:21-22, 2003

Iron Overload Syndromes Hemojuvelin (HJV) 1q21 Juvenile iron overload Autosomal recessive Parenchymal cell distribution Pedigree described Fleming and Bacon, NEJM 352:1741-1744, 2005

Hereditary Hemochromatosis Genetic testing for non-HFE HH InVitae, San Francisco Genetic Testing Company HFE HAMP – hepcidin HFE-2 – hemojuvelin SLC40A1 – ferroportin TFR2 $1500 per order, 2 weeks 415-374-7782 clinical@invitae.com

Hereditary Hemochromatosis Summary In 2013… Most patients with hemochromatosis do not need a liver biopsy Only about 60% of C282Y homozygous patients have phenotypic expression About 25% of C282Y homozygous men have signs or symptoms of hemochromatosis Detailed gene testing available