Hereditary Hemochromatosis

Erin CurrinDecember 11, 2015

Faculty Sponsor: Dr. Richard

What is Iron? • Atomic Number = 26

o 26 electrons, 26 protons, 30 neutrons• Transition Metal• Ductile and Malleable• Conducts Heat and Electricity• Produces magnetic field• Makes up earths core entirely, mantle, crust• Oxidation-Reduction• Important in humans too!

Importance of Iron In Vivo

• Hemoglobino Part of porphyrin ring of heme moleculeo Each of the 4 globin subunit contains 1 iron atomo 2 million erythrocytes created per secondo Each mature erythrocyte: ~ 280 million molecules of hemoglobino Total Iron Flux: 2-3 x 1015 iron atoms/second

• Cytochrome p450• Myoglobin• DNA Synthesis• Mitochondrial function

Drakesmith et al, Cell Metabolsim, 2015; Rishi, Biosci Rep, 2015

Hemoglobin

Iron Redox• Readily undergoes oxidation-reduction reactions

o Ferric iron Fe3+ (oxidized) and ferrous Fe2+ (reduced)o Reduced form- most cytoplastmic irono Fenton Reaction: Donation of electron free radicals

o Free radicals: result in peroxidation of lipids, oxidative damage of DNA and macromolecules

Mackenzie, Antiox and Redox, 2008

Dietary Iron• Diet: the only source of iron • Normal diet: 10-20 mg per day• Normal absorption ~ 1-2 mg, in HH 8-10 mg• Main absorbable iron forms:

o Heme Iron: meat, fisho Non-heme iron: cocoa, lentils, spinach, etc.

• Vitamin C: Co-factor in non-heme iron absorption• Absorption site: Duodenum

o Via apical iron transporter DMT1 -divalent metal transporter 1o 5x more efficient for heme than non-heme irono ? Heme Carrier Protein (HCP1) – possibly low affinity heme- Fe

uptake

Yun, Crit Review in Onc/Heme, 2015; Cook Am J Clin Nutr, 2001; LeBlanc, Am J Cell Physiol, 2012

Duodenal Enterocyte

http://courses.washington.edu/conj/bess/iron/iron.htm; accessed Dec 2015

Iron Ins and Outs

• Total body iron ~ 3-4 gmo 2-3 gm erythrocytes o 1 gm hepatocyteso 2-3 mg in plasma, transferrin-bound

• Require~ 20-25 mg iron dailyo 80% erythropoiesis in the bone marrow ~ 20 mg

• Where does this come from?o 18-19 mg recycled: breakdown of hgb from senescent

RBCs by reticuloentdothelial macrophageso 1-2 mg absorbed by duodenal enterocytes (0.1%)

• Losses: Sloughing of enterocytes 1-2 mg, menses

Pietrangelo, NEJM, 2004; Yun, Crit Rev Onc, 2015; Brissot, Journal of Hepatology, 2015; Ganz, Biochemica, 2012

Pietrangelo, NEJM, 2004

Iron Transport in PlasmaTransferrin

• Absorbed by duodenum enters plasma• Not soluble in plasma (as a metal) • Binds Transferrin (Tf)

o 679 amino acid proteino Produced by hepatocyteso Tf binds 2 iron ions – ferric form-- reversiblyo Delivery to cells (ie: erytrhoid precursors in cells)o Tf-iron complex binds to Tf receptors (TfR1)o transferrin saturation (TS) of approximately 30%

Brissot, Journal of Hepatology, 2015; Keel, Exp Hematology, 2015s

Non‐Transferrin Bound IronLabile Plasma Iron

• Non-transferrin bound iron (NTBI): TS > 45%o Likely non-protein bound and protein boundo Targets parenchymal cellso Especially liver, pancreas, heart

• Labile Plasma Iron (LPI): TS > 75%o Generates ROSo Unregulated transport across cell membraneso May Downregulate of TfRo Redox active, chelatableo Permeates into organso Peroxidation of membrane lipids

Cabantchik, Best Practice & Research Haematology, 2005; Brissot, J Hepatol, 2015

Brissot, Biochima, 2012

NTBI/LPI in HH• Described in non-HH causes of IO• N=159 patients

o 23 patients with IO and HHo 24 patients with iron depleted HHo 33 alcoholic cirrhosiso 63 healthy controls

• NTBI/LPI higher in IO and HH• LPI correlated with abnormal LFTs• LPI present when TS > 75% regardless of cause

Le Lan, Blood 2005

Iron StorageFerritin

• Ferritin major storage molecule• Stores up to 4500 iron atoms• Iron stored as Ferric Iron Fe3+

• Protects cells from free radical damage• Downregulated in iron deficiency• Upregulated in iron overload• Main storage sites:

o Livero Reticuloendothelial cells (macrophage, monocytes)

• Mean Value:o Men 12-300 ng/ml; women 12-150 ng/ml

Brissot, Journal of Hepatology, 2015; Hentz, Cell, 2004 ; Yun, Crit reviews in Onc/Heme, 2015; Medline 2015

Iron ExportFerroportin

• Multidomain transmembrane protein • Encoded by SLC40A1• Sole Iron Exporter

o Basolateral membrane of duodenal enterocyteo Plasma membrane of macrophage (splenic, hepatic)o Hepatocytes, Placental Syncytiotrophoblastso Lung, renal tubules, erythrocyte precursors

• Ferrportin in iron deficiencyo Enhances iron export into plasma

• Regulated by the protein hepcidin

Ganz, Hematology, 2011; Ganz, Biochemica, 2012; Pietrangelo, Gastroenterology, 2015; Cianetti, Advances in Hematology, 2010

Regulator of Ferroportin: Hepcidin

• Hepcidin 25 amino acid protein• Mainly produced by hepatocytes• Major regulator of plasma iron• Encoded by HAMP• Function:

o Hepcidin binds to ferroportino Internalization and destruction of ferroportino Decreases cellular iron export o Inhibits dietary absorption, macrophage iron recycling

• … what controls hepcidin?Ganz, Hematology, 2011; Bissot, Journal of Hepatology, 2015

Hepcidin‐FerroportinSystem

Rishi, Blood, 2015

HepcidinRegulation

Regulators of Hepcidin1) Inflammation/Infection

o Inflammatory signals, ie: IL-6 by monocytes/macrophages via TLRs during infection

o TNF alpha, IFN gammao Lipopolysaccharide (LPS)

2) Hypoxiao Hypoxic conditions may decrease production of HAMP

Nicolas, J Clin Invest, 2002; Rishi,Biosci, 2015 

Regulators of Hepcidincont.

3) Erythroid Regulators: Several potential regulators• Discovery of hormone erythroferone (EFRE)

o Thought to meditate hepcidin during stress hematopoiesis• EFRE knockout mice failed to suppress hepcidin• Delayed recovery from hemorrhage

o Mice with thalessemia intermedia– increase EFRE• May contributes to suppression of hepcidin

o Uncertain role in homeostasis

• TFRCo TFEC peak level on proerythroblastso TFRC expression on erythroids may suppress hepcidino Via soluble factor Xo TFR1 may increase ERFE expression

Kautz et al, Nature Genetics, 2014;  Koury, The Hematologist, 2015l Keel Exp Hematology, 2015

Regulators of Hepcidincont.

4) Iron StoresHFE gene: modulator of hepcidin

• Feder et al, 1996 Identified a MHC Class 1 like gene, HFE• Does not participate in antigen presentation• Mutated in HH

• 343 amino acid protein• 3 extracellular domains

Barton et al, Gene, 2015; Feder et al, 1996, 

HFE Mutations3 missense mutations: missense

1) C282Y • Tyrosine replace

cysteine at position 2822) H63D

• Aspartic acid replaces histadine at position 63

3) S65C (later discovery)• Cysteine replace serine

at position 65Mutations result in decreased hepcidin

Feder, Nature, 1996; Mura, Blood, 1999

S65C

How does HFE regulate Hepcidin?

• HFE thought to complex with TFRCo Transgenic induction of hepcidin without TFRC

• BMP (bone morphogenic protein) pathway in livero Activated BMP-1 receptors phosphorylate SMAD proteinso In turn, induce hepcidin transcription

• Wu et al, 2014• Wild type HFE binds to BMP -1 Receptor “Alk 3”

o Cultured hepatocytes and mouse livero HFE increase Alk 3 expression on cell surfaceo Activates BMP/SMAD signaling hepcidin transcription

Wu, Blood, 2014; Muckenthaler, Blood, 2014

Muckenthaler, Blood, 2014

HFEmutations and hepcidin

• C282Y and H63D • Mutations fail to increase Alk3 on cell surface• Impairment of BMP mediated activation pathway• Mechanisms differ:

o Speculated that HFE C282Y does not reach the cell surface • Sequesters Alk3 within the cell

o H63D failed to inhibit ubiquitination of ALK3• Further work needed

HFE Hereditary Hemochromatosis

HemochromatosisHistory

• Recognized in 1880so Diabetes, bronze skin and cirrhosiso 1889: Term Hemochromatosis for bronze stained organs

• 1935: disease recognized as inheritedo Metabolic defect causing excess iron in tissues

• 1970s-80s: autosomal recessiveo Short arm of chromosome 6, encoding HLA-A*3

• 1996: “hemochromatosis gene” (HFE) identified• C282Y• H63D

Pietrangelo, NEJM, 2004Simon, Pawlotsky, et al, Nouv Presse Med, 1975; Simon, Le Mignon et al, Am J Hum Genet 1987

Pathology Education Informational Resource (PEIR) Digital Library

HFE: Many new mutations discovered

Barton et al, Gene, 2015

Types of HHType Pathogeneic Mutations Mutation

FrequencyOnset < 30 years

1) Classical HFE: C282Y, H63D, S65C, others C282Y, H63D Common

AR Uncommon,

2) Non Classical “Juvenile”

2A: mutations in HAMP 2B: HJV Hemojuvelin *

Rare AR Common

3) NonClassical

TFR-2 Transferrin Receptor Protein 2

Rare AR Common

4) Non Classical“FerroportinDisease”

SLC40A1Ferroportin

Q248Hcommon in SubsaharanAfrica

AD Uncommon

Crownover, AFP, 2013; Zanella, Blood, 2015; Pietrangelo, NEJM, 2004

AR= autosomal recessive, AD = autosomal dominan* HJV: binds to BMP proteins

C282Y Prevalence• Most common form of HH: 80-85% patients• Arise from Celtic/Viking ancestor ~ 4000 years ago• Does not affect reproduction• Protective? • Mean allelic frequency ~ 6% Caucasians• C282Y homozygosity in Caucasians: 1:200-300

o Geographic variation, migrationo As high as 1:83 Irelando 10x more common than CF dF508 gene

• H63D mean allelic frequency 14% Caucasians

Pietrangelo, Gastroenterology, 2015; Adams PC, Lancet, 2007; Bacon, Hepatology, 2011

Single Allele FrequencyC282Y H63D

Distante, Human Gen, 2004

Prevalence of C282Y 30, 672 caucasian subjects Kaiser Permanante, CA

Heterozygous0.937 %

Homozygous0.062%

Beutler, Blood, 2003

Genotypic and Phenotypic Profiles

• n=123, referred for elevated TS, asymptomatic• Genotyped, phlebotomy to iron depletion• mobilized Iron quantified (gm Iron = hct/3x wt of blood x .0035)

Sham et al, Blood, 2000

Varied Iron Loading

Waalen, Best Practice & Research Clin Haem, 2005

Normal Ferritin in C282Y homozygotes

Bacon, AASLD, Hepatology, 2011

HealthIron Study

• N=31,192 persons N. European/Australian descent• age 40-69, prospective• 203 patients with HH, followed avg 12 years• Cirrhosis, fibrosis, HCC, abnormal LFTs, 2-3rd MCPs• 28.4% Men, 1.2% women• Ferritin > 1000ug/L more cirrhosis, more symptoms• Arthropathy unrelated to ferritin levels

Allen, NEJM, 2008

ScreeningRecommendations

• Per AASLD practice guidelines:o Patients with suggestive symptomso Characteristic physical findingso Family Historyo All first degree relatives of HH patient

Bacon, Hepatology, 2011

ScreeningRecommendations

• Approach to Diagnosis: Who should get gene testing?o If TS >45% (serum iron/TIBC X 100)

• ~ 45% high sensitivity for detecting C282Y homozygotes• Lower specificity /PPV picks up mild iron overload

o ferritin > normal • 200 women mcg/dl, 300 men ug/L mcg/dl• No difference fasting vs. not fasting• Less biologic variability than TS• High false positive

o If both abnormal, order HFE gene testing

Population Studies• Hemochromatosis and Iron Overload Study (HEIRS)

99,711 North Americanso 88% men C282Y/C282Y had Ferritin >300 ug/L o 57% women C282Y/C282Y Ferritin > 200 Ug/L

• Kaiser Permanente Studyo 77% men C282Y/C282Y Ferritin >250 ug/Lo 56% women C282Y/C282Y Ferritin > 200 ug/L

• Study in Individuals <35 yearso TS <45% and normal ferritin NPV 97%

Bacon, Hepatology, 2011; Beutler, Blood, 2002; Adams, NEJM, 2005

Clinical Manifestations

Disease Presentation before Gene testing

• Symptoms BEFORE era of screening: o Weakness, Lethargyo Abdominal Paino Impotenceo Cardiac Failure

• Physical Findings BEFORE era of screening:o Cirrhosis/HCCo Hepatomegalyo Gynecomastiao Testicular Atrophyo Skin pigmentationo Diabeteso Arthropathy 2nd-3rd MCP

• Classic Triad DM, Cirrhosis, Skin findings > 20 gm Iron

Bacon, Gastroenterology, 2011

Disease Stages in gene testing era

European Assoc Study of Liver Disease Consensus:Stage 1: Refers to patients with genetic disorder with no increase in iron stores who have a genetic susceptibilityStage 2: patients with genetic disorder who have phenotypic evidence of iron overload without tissue or organ damageStage 3: genetic disorder with iron overload and iron deposition with tissue and organ damage occurs

Bacon, Hepatology, 2011; Allen, NEJM, 2008

Bacon, Hepatology, 2011

Liver Manifestations• Excess iron deposition in liver, hepatocytes• Fibrosis, Cirrhosis: ferritin > 1000 ng/ml• HCC develops ~ 8-10% patients with HH

o Cirrhosis almost always presento RR 20, annual risk 3-4% incidence if cirrhosiso Follow guidelines for advanced fibrosis/cirrhosis

• HCC Surveillance if cirrhosiso Every 6 month US, gold standardo Sensitivity 94%

Kew, Liver Cancer, 2014; Crownover, AFP, 2013; Colombo, Uptodate Prevention HCC, 2015

Disease Modifiers

Pietrangelo, Gastroenterology, 2015; Bacon, Hepatology, 2011; Fletcher, Gastroenterology, 2002; Tayrac, J Hepatol, 2015

• ALCOHOL• Australian C282Y/C282Y > 60 gm /day ETOH

o 60% cirrhosis vs. < 7% no alcoholo Alcohol induce hepatic fibrogenesis and oxidative stress

• NAFLD/NASH• Hepatitis B, C• Genetic, Polymorphisms in genes

o FPN, MP, Tf, TFR2, ARNTL, GNPATo Genome Wide Association Study

Rs2811647 Tf polymorphism increased Tf and serum Iron• Male

Cirrhosis rates in HH

Assessment Intrahepatic Iron

• Gold standard-Liver biopsyo Measurement of hepatic Iron concentration (HIC)o > 80 micromoles/gm of dry weighto Hepatic Index > 1.9 mol/kg/yr (HIC/age yrs)o Invasive

• Meta-Analysis of MRI for identifying Iron Overloado 20 studieso Identified patients without iron overload

• Negative LLR 0.10, 0.05o Less accurate for definite diagnosis of iron overload

• Positive LLR 8.85, 4.86

Sarigianni, et al, Clinical Gast and Hep, 2015; Bacon, Clin Gast and Hep, 2015

Liver Imaging• T2* MRI - relaxometry method-weighted MRI

o Very quick, single breath holdo Approximation of stored irono Correlates with iron loadingo Limitations with calibration (only at 1.5T)

• Dynamic elastrography:o Surrogate for fibrosis evaluationo assessment of propagation of shear waves within tissues to

calculate visco-elastic propertieso First Generation: Transient elastrography (FibroScan)o Second Generation: ARF (acoustic radiation force) o Used widely in Europe

Van Beers, Journal of Hepatology, 2015; St. Pierre, Magn Reason Med, 2014; Knovich, Blood Rev, 2009 

FibroScan

Cardiac Manifestations• Usually occurs after significant hepatic iron uptake• Clinical Features:

o Early restrictive cardiomyopathy, diastolic dysfunctiono Progress to end stage dilated cardiomyopathyo Biventricular heart failureo Predisposition to arrhythmias, conduction system, AV block

• Diagnosis:o Biopsy- INVASIVE! o ECHO- can determine cardiac functiono Cardiac MRI: T2* MRI

• if < 20 ms, correlate with cardiac iron loading

Bejar, Clin Med Insights: Cardiology, 2015; Murphy, Journal of Cardiac Failure, 2010

Joint Manifestations• Most common affected joints:

o 2nd/3rd MCPo Wrists, hip, ankleso Worse in women

• Presentation:o OA like symptoms, pseudogout, synovitis resembling RAo Chondrocalcinosis of wrist/knee up to 50% patientso High risk severe large joint requiring joint replacement

• Radiographso Joint space narrowing, subchondral cysts, osteophytes

• Treatmento Often not improved by phlebotomy

Husar‐Memmer, Curr Rheumatol Rep, 2014; Adams, Blood, 2010

Husar‐Memmer, Curr Rheumatol Rep, 2014

Other Manifestations• DM

o Occurs late in diseaseo Most patients with DM already have cirrhosis

• Hypopituitarismo Iron deposition in pituitary cells – anterior > posterioro Decreased levels of trophic hormones

• Testicular Atrophyo Secondary from pituitary insufficiencyo Less commonly testicular iron deposition

• Skin pigmentation

Treatment

Treatment: Phlebotomy• Treatment of choice for HH: Phlebotomy• No randomized trial• Starting phlebotomy before cirrhosis/DM

o Goal: prevention of end organ damageo Large population based studies show decreased survival in

untreated HH patients• One unit of blood: 200-250 mg iron

o When IO severe, > 30 gm, ~ 2-3 years to removeo Check hct/hgb prior to phlebotomyo Avoid l < 80% starting hcto Ferritin falls faster iron mobilization, reflects depleted storeso TS can remain elevated until stores are depleted

Bacon, Hepatology, 2015; Adams, Blood, 2010; Adams, Gastroenterology, 1991

Treatment Algorithm

Adams, Blood, 2010 

Treatment and Maintenance

• Goal: Serum Ferritin of 50 ug/L or lesso Some practitioners and patients may target ~ 20-30 ug/Lo SF preferred over TS o TS may remain high when body iron stores when SF lowo Targeting TS may result in iron deficiency, fatigue

• Rate of re-accumulation o Wide variationo Some require monthly maintenance, others 1-2x/yearo Others may go yearso Check SF every 6 months, personalized maintenance plan

Phlebotomy‐ increase Iron absorption?

• Lynch et al, Blood, 1989• Measured iron absorption from test meals in HH

Serum Ferritin Non Heme Iron Absorption

Heme Iron Absorption

Untreated: SF>1000 ug/L

9% 41%

Partially treatedSF ~ 538 ug/L

12% 50%

Fully TreatedSF ~ 14 ug/L

42% 39%

Lynch, Blood, 1989, Adams, Blood, 2010; 

Reversal of liver damage• Falize et al, 2006• 36 patients with C282Y/C282Y

o 13 patients with Grade 3 fibrosis, 23 patients with Grade 4 o 2 biopsies, lag time of 2 years, treatment with phlebotomyo Mean follow up of 9.7 yearso Fibrosis regression correlated with compliance to therapy

69%35%

Reversal of other end‐organ damage

• Cardiac:o LV dysfunction improved with phlebotomyo Dilated cardiomyopathy likely not reversedo Fluid shifts with phlebotomy maybe challenging with CHF

• Diabeteso Improved glucose control, DM usually not reversed

• Skino decreased pigmentation

• Livero Reduction in LFTso Fibrosis can be reversedo Reduction in HCC risk if iron removed before cirrhosis

• Testicular Atrophyo No reversal

Intolerance of Phlebotomy• Bone marrow unable to recover• End organ damage, ie: CHF, unable to tolerate

volume shifts, no IV access• Options

o Erythrocytapheresiso Chelating agents

• Deferoxamine• Deferasirox

Therapeutic Erythrocytapharesis

• Therapeutic Erythrocytopharesis (TE)• Removes iron as hemoglobin

o must have no or mild anemiao must have sufficient rate of effective erythropoiesis

• Isovolemic, large volume TE removes more blood per session than phlebotomyo Spares plasma proteins, coagulation factors, proteinso May achieve iron depletion more quicklyo More costly than phlebotomy

• Side effects: o transient hypovolemia, monitor iron levels closely

• Rarely used

Adams, Blood, 2010

Deferoxamine• Desferal• Subcutaneous Infusion• Urine as chelate• Most experience in non-HH patients• Small case series, DFO as effective as phlebotomy

500 ml/weekly in removing irono Cardiomyopathy improved in 1 patient

• Compliance- fair• Expensive• Side Effects: infusion site reactions, hearing, vision,

growth, skeletal abnormalities, zinc deficiency, Yersinia infection, renal effects

Nielsen, British Journal of Hematology, 2003; Adams, Blood, 2010

Deferasirox• Exjade• Oral• Stool as chelate• Phase 1/2 Dose escalation Trial, 48 week (2010)

o C282Y/C282Y; SF 200-3000 ng/ml, TS > 45%, no cirrhosis, n=49o Starting dose of 5 mg/kg; 10 mg/kg; 15 mg/kgo Side effects dose dependento LFTs abnormalities (6) and increased Cr (8)o Diarrhea, nausea, abdominal paino Mean SF decrease after 48 weeks respectively: 63%, 74%, 74% o Dose of 10 mg/kg/day appears safest

• SE: renal failure, hepatic failure, GI hemorrhage• Expensive

Adams, Blood, 2010; Phatak, Hepatology, 2010

Other Therapeutic Options

• Deferiprone (Ferriprox)o Could not find data for use in HFE HH

• Proton Pump Inhibitors:o Reduced absorption of non-heme iron in test meal (7 patients)

• Insufficient data to make recommendation

Hutchinson, Gut, 2007; Adams, Blood, 2010

Before PPI

After PPI for 7 day

Dietary Management

• Black tea inhibits absorption non-heme iron• Vibrio Vulnificans– warm coastal waters, GN Bacillus

Adams, Blood, 2010