Emocromatosi ed Iperferritinemie Alberto Piperno Università di Milano-Bicocca Dipartimento di Medicina e Chirurgia Centro Malattie Rare ASST-Monza, Ospedale S.Gerardo Learning Objectives

• Cos’è l’emocromatosi • Cos’è l’iperferritinemia • La diagnosi di emocromatosi • L’intrico diagnostico delle iperferritinemie • Take home messages Emocromatosi Iperferritinemia

Un gruppo di malattie Può essere espressione di: ereditarie caratterizzate da: • accumulo di ferro da cause • elevata saturazione della diverse (genetiche e transferrina acquisite) • iperferritinemia • accumulo di ferro di entità Più frequentemente è dovuta a: variabile • diverse condizioni (genetiche • sviluppo di complicanze con e acquisite) non associate ad variabile penetranza ed un corrispondente incremento espressione dei depositi di ferro Hereditary Hemochromatosis diseases, genes and proteins

Disease Gene Protein Hemochromatosis type 1 HFE HFE Hemochromatosis type 2a (JH) HAMP Hepcidin Hemochromatosis type 2b (JH) HJV Hemojuvelin Hemochromatosis type 3 TFR2 Transferrin receptor 2 Hemochromatosis type 4 SLC40A1 Ferroportin

Gene Phenotype Allele frequency 1000G ESP6500 ExAC

Recessive

Recessive

Recessive Recessive Dominant

Wallace & Subramaniam Genet Med 2016;18:618-626; Piperno A. Expert Opin. Med. Diagn. 2013;7:161-177; Pietrangelo A. Hepatology 2007;,46::1291-1301; Piperno et al doi: 10.21037/tgh.2019.11.15 Hemochromatosis proteins and iron homeostasis

Blood and tissue iron status

LSECs I II

TF-Fe - - 2 BMP2 BMP6 Erythropoietic TFR2 BMPR BMPR HFE HJV HIF

IL-6 TFR1 Hypoxia

IL-6R TMPRSS6 PDGF-BB

ERK1/2? activity

Inflammation SMAD4 ERFE STAT3 Nucleus ? Hepatocyte HAMP Duodenal Macrophage cell Hepcidin Ferroportin Ferroportin Iron release in blood

Modified from Expert Opin Med Diagn 2013; Silvestri et al 2019; Tangudu et al 2019 Hemochromatosis type 1, 2 and 3 a quantitative defect of hepcidin production

HFE HJV-HAMP TFR2

Hepcidin Inadequate production Reduced production

Adult Juvenile Type 3 Hemochromatosis Hemochromatosis Hemochromatosis

120 100

epcidin 80 h 60 40

/mg creatinine) /mg 20 urinary

ng 0 Controls HFE TfR2 HFE2 HAMP (

Piperno et al WJG 2009;15:513 HH type-4: gain-of-function mutations of SLC40A1 gene causes hepcidin resistence

• Genetics: autosomal dominant; gain-of-function mutations; incomplete penetrance and variable expression at biochemical and clinical level • Clinics: similar to hemochromatosis type-1 • Laboratory: variably increased TSAT and serum ferritin

Hepcidin resistence 30-year-old men Tf saturation 88%; SF 5600 µg/L; macrophage LIC 14.5 mg Fe/g dry wt Fe Enterocyte RBC RBC

Cp Mut FP FP FP Mut FP Heph High Fe High TS Hepatocellular IOL

Pietrangelo et al. N Engl J Med 1999; Pietrangelo A Haematologica 2017; Le Lan et al. Gastroenterology 2011 Physiopathology of iron overload in Hemochromatosis

Type 1, 2, 3, and 4 HH: increased iron absorption and macrophage release

§ High serum iron RES involvement § Increased Tf saturation (TSAT) RES occurs in advanced § Formation of NTBI in the plasma liver IOL and favours fibrosis

Hepatocellular IOL §High serum ferritin Excess iron overwhelms hepatocyte capacity Diabetes Hypogonadism Cardiopathy Liver damage Systemic IOL Arthropathy

NTBI, non-transferrin-bound iron; RES, reticuloendothelial system; Tf, transferrin. Transl Gastroenterol Hepatol 2020;5:25 Ferritin: the iron storage protein

• In mammalian ferritins, 24 subunits assemble to form a hollow symmetrical protein with a molecular weight of about 480 kDa. The cavity contains up to 4,500 iron atoms. • Three different types of monomer are in mammalian cells: – H (FtH, heavy, ~21 kDa) and L (FtL, light,~19 kDa) monomers variably assemble to form the most abundant cytosolic ferritins (isoferritins). – The mitochondrial subunit (FtMt, ~21 kDa) is found exclusively in the mitochondria (FtMt).

Arch Toxicol (2014) 88:1787–1802 Tissue and serum ferritin

Serum ferritin in humans ferritin appears to consist of l- L-ferritin ferritin, a glycosylated Iron leakage L-ferritin form of L termed G- ferritin, a truncated ferritin G-ferritin (smaller) form of L (S- S-ferritin ferritin) and trace secretion H-ferritin amounts of H-ferritin. Cytokines Serum ferritin is iron- poor compared to intracellular ferritin Hepatocytes, Macrophages, Myocytes, other cells Cell Serum

Metallomics 2014;6:748; Arch Toxicol 2014;88:1787; Blood. 2010;116:1574-84. Physiopathology of hyperferritinemia

Iron overload Cell necrosis

L-ferritin ferritin Iron leakage L-ferritin G-ferritin ferritin S-ferritin secretion H-ferritin Cytokines

Inflammation Synthesis Altered/variable Altered/variable Metabolic dis. dysregulation secretion? scavenging? Hypertyroidism

Cell Serum

Arch Toxicol (2014) 88:1787–1802; J Biol Chem 2020;295:15727; J Neural Transm (2011) 118:337–347 Hyperferritinemia is not a reliable index of iron overload

Serum ferritin as an index of iron overload shows marked variability according to different clinical situations

Olynik et al Clin Gastroenterol Hepatol 2009 Hyperferritinemia is not a reliable index of iron overload

No iron overload Systemic iron overload Acquired Inflammation Primary iron overload Hepatocellular necrosis Hemochromatosis (type 1, 2 and 3) Metabolic disorders (DHF) Hemochromatosis type 4 High alcohol intake Hyperthyroidism Secondary iron overload Inherited Iron-loading anemias Hereditary hyperferritinemia-cataract Transfusional iron overload Benign hyperferritinemia End-stage liver disease Chronic liver diseases Local iron overload [Mostly mild to moderate with prevalent Other iron overload diseases reticuloendothelial or mixed distribution] Acquired Acquired Neonatal hemochromatosis (GALD) Chronic liver diseases (viral, ASH, NASH) African iron overload Metabolic disorders (DIOS) Inherited Inherited DMT1 deficiency Ferroportin disease Hypotransferrinemia Gaucher’s disease Aceruloplasminemia

Modified from Expert Opin. Med. Diagn. 2013;7:161; Transl Gastroenterol Hepatol 2020;5:25 Hyperferritinemia should be classified according to transferrin saturation

<45% Transferrin saturation

No iron overload Inflammatory tests Inflammation Liver function tests Hepatocellular necrosis Glucose, Lipid tests, OGTT Metabolic disorders (DHF) History High alcohol intake Familial and personal history of early cataract, Hereditary hyperferritinemia-cataract genetic test Benign hyperferritinemia Exclusion diagnosis, genetic test

Iron overload (mostly mild to moderate) Chronic liver diseases (viral, ASH, NASH) Liver function tests Metabolic disorders (DIOS) Glucose, Lipid tests, OGTT Hemolytic anemias Hemolytic tests Transfusional iron overload (early stages) History, number of transfusions Parenteral iron administration (inadequate) History, number of inadequate infusion Ferroportin disease Family recurrence, genetic test Aceruloplasminemia Measure serum ceruloplasmin

Gaucher’s disease Platelet, splenomegaly, biochemical test

Modified from Expert Opin. Med. Diagn. 2013;7:161-177; Transl Gastroenterol Hepatol 2020;5:25 Causes of hyperferritinemia

macrophage 2+ Transfusion-dependent: Fp Fe Early RES iron loading -> Cp Fe2+ hyperferritinemia with 3+ Tf-Fe TfR1 hepatocyte normal TSAT; Fp Cp Fe3+ Later TSAT oversaturation -> systemic parenchimal TF > TSAT iron overload

enterocytes RES Iron retention: macrophage Inflammation (cytokine 2+ mediated: hepcidin and Fe Fe2+ GR Cp Fp ferritin up-regulation) Fp GR Heph Ferroportin disease (Loss- GR of-function mutations) Tf Hyperferritinemia with reduced or normal TSAT Metabolic-associated hyperferritinemia, and iron overload: a pathologic continuum

Metabolic syndrome

DIOS Insulin

Local Altered hepcidin inflammation NAFLD production? Hepatic damage

Hepatic and Sinusoidal iron Serum ferritin cellular iron retention? Genetic background Hepcidin-mediated

J Hepatol 2007;46:549–552 Causes of Hyperferritinemia: aceruloplasminemia

Apo-Ceruloplasmin Iron accumulation 6-Cu Brain

Ceruloplasmin Iron accumulation Ferroportin High s-ferritin liver, pancreas Fe3+ Transferrin

Low serum iron, Increased iron Low transferrin saturation absorption

Stomach Iron deficient erythropoiesis Hepcidin Duodenum suppression Mild microcytic anemia Hyperferritinemia should be classified according to transferrin saturation

Transferrin saturation >45%

Primary iron overload Genetic tests Hemochromatosis (type 1, 2 and 3) Hemochromatosis type 4

Secondary iron overload Inherited/acquired ineffective erythropoiesis: Iron-loading anemias anemia, bone marrow aspiration/biopsy, genetics End-stage liver disease Liver function and instrumental tests

History, number of transfusions Transfusional iron overload (later stages)

Other iron overload diseases Severe microcytic anemia; measure serum DMT1 deficiency transferrin; Genetic tests Hypotransferrinemia

Severe liver failure Neonatal hemochromatosis

African iron overload

Modified from Expert Opin. Med. Diagn. 2013;7:161-177 Diagnosis of hemochromatosis (and other iron overload disorders)

• Define the presence and the amount of iron overload • Genetic testing if appropriate • Define appropriate tests for clinical stadiation (when and how) • Define adequate therapy • Define adequate follow-up Defining and monitoring the amount of iron overload

Biochemical indices § Transferrin saturation (n.v. 16-45%) Qualitative, not quantitative index [s-Iron (µg/dL)/s-Transferrin (mg/dL)*1.42] § Serum ferritin (n.v. varies according Correlates with body iron, but to sex and age: children 12/30-100 influenced by many factors ng/mL; young women 12/30-160 ng/mL; post-menopausal women 12/30-250 ng/mL; men 12/30-400)

Non-invasive methods Reliable, can be applied to different § Quantitative MRI (diagnostic) organs § SQUID (diagnostic) Reliable for liver; not easily available

Liver biopsy Gold standard for LIC and evaluation of iron distribution, liver damage and associated pathologies; invasive and not repeatable; can be replaced by non invasive methods (Fibroscan). Defining and monitoring organ dysfunction

• Heart functions (cardiac ultrasound, MRI) • Endocrine dysfunction (lab. Tests) • Liver dysfunction (lab. Tests, abdominal ultrasound, TC, MRI and elastography)

PDTA regione Lombardia Genetic testing in hemochromatosis

HFE HFE-HH [C282Y/C282Y] has variable penetrance C282Y/C282Y 100% and expression that is C282Y/H63D C282Y/rare much lower in C282Y/H63D H63D/H63D HFE deletion and H63D/H63D

75% Biochemical penetrance

50%

Clinical penetrance 25%

Gastroenterology 2000;119:441-5; J Med Genet 2004;41:721; Expert Opin. Med. Diagn. 2013;7:161; Transl Gastroenterol Hepatol 2020;5:25 Flow-chart 1 Saturazione della transferrina >45% Valutare cause secondarie: Percorso diagnostico terapeutico Ferritina aumentata rispetto all’atteso -epatopatia cronica di vari eziologia dei casi con saturazione della per età e sesso -anemie associate a sovraccarico di transferrina e ferritina elevate ferro

PDTA – Regione Lombardia Conferma del dato

Test genetico HFE:mutazioni C282Y e H63D

Omozigosi C282Y Eterozigosi C282Y, H63D, wild type (eterozigosi C282Y/H63D, omozigosi H63D*)

ferritina ferritina ferritina <300 µg/L 300-1000 µg/L >1000µg/L ferritina ferritina ferritina <300µg/L 300-1000µg/L >1000µg/L

RM quantitativa e/o biopsia epatica e studio danno d’organo**

Controllo a Valutazione Biopsia epatica** Controllo a Sovraccarico di ferro un anno danno d’organo e studio del un anno danno d’organo SI NO

Follow up Terapia Follow up Ulteriori test***: Riconsidera HFE, TfR2, HJV, cause HAMP, SLC40A1 (tabella 3)

* l’eterozigosi C282Y/H63D e soprattutto l’omozigosi H63D hanno bassa penetranza ed espressività e vanno valutate con senso critico (vedi testo). Positivo Negativo ** la biopsia epatica è spesso necessaria a scopo prognostico per valori di ferritina >1000 µg/L o per valori inferiori ma in presenza di cofattori di danno epatico. A scopo diagnostico può essere utile nelle forme di emocromatosi non-HFE. Emocromatosi Emocromatosi non *** la tempistica con cui eseguire tali indagini può variare in base ad alcune tipo 1,2 3,4 determinata caratteristiche cliniche e genetiche (età, sesso, entità del sovraccarico di ferro, distribuzione del ferro epatico, familiarità dominante, presenza di cofattori di danno epatico) Terapia Genetic testing in rare forms of hemochromatosis

HFE rare mutations HJV (type 2) HAMP (type 2) TFR2 (type 3) SLC40A1 (type 4) Other?

Often private mutations Need gene sequencing

Sanger step-by-step NGS panel

Gastroenterology 2000;119:441-5; J Med Genet 2004;41:721; Expert Opin. Med. Diagn. 2013;7:161; Transl Gastroenterol Hepatol 2020;5:25 The diagnostic maze of hyperferritinemia

• First evaluate transferrin saturation • Evaluate alternative causes of hyperferritinemia unrelated to iron overload and correct them if possible • Quantifies the iron overload if needed (MRI) • Perform family study if needed and genetic testing if appropriate • Define adequate therapy • Define adequate follow-up Flow-chart 2 Ferritina aumentata rispetto all’atteso escludere/valutare forme secondarie : Percorso diagnostico terapeutico per età e sesso - epatopatie croniche inclusa NAFLD dei casi con iperferritinemia - alterazioni metaboliche - stato infiammatorio isolata (saturazione della Saturazione della transferrina: <45% - emotrasfusioni/infusioni di ferro ev transferrina normale)

Considera: HHCS [dominante] Ferritina*: <400 µg/L donna Ferritina: Ferritina: Iperferritinemia benigna <600 µg/L uomo >400-600 µg/L <1000 µg/L >1000µg/L [dominante] Iperferritinemia benigna [recessiva] Gaucher [recessiva]

Controllo a RM quantitativa e/o biopsia epatica e studio del un anno danno d’organo** Studio familiare

follow up Sovraccarico tissutale Sequenza IRE L-ferritina Sequenza L-ferritina Analisi enzimatica b-GBA SI NO

familiarità dominante Riconsidera le cause secondarie sequenza SLC40A1 HFE***

terapia

PDTA regione Lombardia Modified from Transl Gastroenterol Hepatol 2020;5:25

* questi valori sono arbitrari perché non esistono dati sufficienti per stabilire un valore soglia della ferritina sopra il quale sia opportuno procedere verso indagini ulteriori. I valori proposti si basano sull’esperienza clinica e riguardano soggetti adulti. ** la biopsia epatica è spesso necessaria a scopo prognostico per valori di ferritina >1000 µg/L e talvolta a scopo diagnostico (per es.: nelle forme di emocromatosi non-HFE). *** lo stato di eterozigosi composta C282Y/H63D, omozigosi H63D e stati di eterozigosi composta tra mutazioni comuni e mutazioni rare lievi (per es.:S65C) possono determinare un fenotipo biochimico caratterizzato da iperferritinemia e saturazione della transferrina al di sotto del valore soglia di 45%. Take home messages

• Iperferritinemia non è sinonimo di sovraccarico di ferro né tantomeno di emocromatosi. • La diagnosi di emocromatosi va sospettata solo nei casi in cui l’iperferritinemia si accompagni ad un’elevata e confermata saturazione della transferrina. • Le cause di iperferritinemia e di sovraccarico di ferro sono numerose ed è necessaria una valutazione clinica accurata a vari livelli. • Sebbene la ferritina sierica sia considerata un indice di sovraccarico di ferro, in molti casi è solo un indice sensibile di varie problematiche tra cui le più comuni sono le patologie epatiche croniche, metaboliche e infiammatorie. • In questi casi la sua unica utilità è quella di permettere di evidenziare le patologie responsabili e avviare un programma terapeutico adeguato verificato attraverso i marker più specifici coerenti con le problematiche in essere