hemochromatosis bcsls telehealth presentation october 19, 2006 gillian lockitch, mbchb, md, frcpc

Hemochromatosis

BCSLS Telehealth PresentationOctober 19, 2006

Gillian Lockitch, MBChB, MD, FRCPC

OBJECTIVES

• Review iron absorption and transport

• Describe types of hemochromatosis

• Hemochromatosis and the laboratory – Suspicion– Investigation – Diagnosis

Iron Absorption and

Transport

Transferrin

FeFe

Macrophage

Fe Fe

Villusenterocyte

Ferritin

Fe+3 Fe+2

Fe2+

Fe+2

Fe+3

Fe+3Fe+2

Liver

Bone Marrow

Ceruloplasmin

Normal Iron metabolism

• 3 - 4 g

• 1 – 2 mg

• 1- 2 mg

• Total body iron

• Daily iron absorption

• Daily iron lossOTHER

• Menstrual (monthly)

• pregnancy (total)

• 2 - 3 g

• 1 – 2 mg

• 1- 2 mgOTHER

• 20 - 40 mg• 600 - 900 mg

Male Female

Fe Fe

Villusenterocyte

Fe+3 Fe+2

Fe2+

Ferritin

Liver

Bone Marrow

20 mg / d

Macrophage

Menstruation20 – 40 mg /m

1 – 2 mg /d

1 – 2 mg /d

Fe Fe

Villusenterocyte

Fe+3 Fe+2

Fe2+

Ferritin

Liver

Bone Marrow

20 mg / d

Macrophage

Menstruation20 – 40 mg /m

7 – 8 mg /d

1 – 2 mg /d

Iron transport and absorption proteins

• Transferrin

• Ferritin

• Transferrin receptor

• Ceruloplasmin

Non-heme iron absorption process

• Reduction of ferric to ferrous iron• Transport across brush border• Sequestration in enterocyte• Basal transport from cell• Oxidation to ferric form• Transport by transferrin • Uptake by transferrin receptors• Utilisation or storage

Ferric Ferric

reductasereductase

FerroportinFerroportin(IReg1)(IReg1)

DMT1DMT1

HephaestinHephaestin

Fe Fe 2+2+

Fe Fe 2+2+Fe Fe 3+3+

Fe Fe 2+2+

Fe Fe 3+3+

Fe Fe 2+2+

Reduction of ferric to ferrous iron

Transport across brush border

Sequestration in enterocyte

Oxidation to ferric form

Basal transport from cell

Ferric reductaseFerric reductase

Divalent metal transporter 1Divalent metal transporter 1(DMT1)(DMT1)

FerritinFerritin

FerroportinFerroportin

HephaestinHephaestinCeruloplasminCeruloplasmin

Iron responsive elements (IREs)

Iron responsive element binding proteins (IRPs)

Iron regulates the synthesis of its own key

transport and storage proteins

Protein

DNA

Messenger RNA

transcription

translation

5`5` 3`3`

Untranslated regionsUntranslated regions

mRNA transcriptmRNA transcript IREIRE

Iron Responsive Elements (IREs)Iron Responsive Elements (IREs)

IREIRE

Low intracellular iron: IRP binds and prevents mRNA translation

Low intracellular iron: IRP binds and stabilizes mRNA

IRP1 and IRP2 - Binding ProteinsIRP1 and IRP2 - Binding Proteins

Proteins with 5’ or 3’ IREs

• 5’ - low iron decreases synthesis– Ferritin– Ferroportin– Erythroid heme aminolevulinic acid synthase

• 3’ - low iron prevents mRNA degradation– Transferrin receptor 1– DMT1 (divalent metal transporter)

FerritinFerritin

mRNA transcriptmRNA transcript

5`5`

Low intracellular iron Low intracellular iron contentcontentIRP boundIRP bound

mRNA not translatedmRNA not translatedferritin lowferritin low

High intracellular iron contentHigh intracellular iron contentIRP not boundIRP not boundmRNA translatedmRNA translatedferritin synthesizedferritin synthesized

DMT1DMT1

mRNA transcriptmRNA transcript

RTDRTD 3`3`

IREsIREs

Control of synthesisControl of synthesis

Low intracellular iron contentLow intracellular iron content

IRPs bound - mRNA stabilizedIRPs bound - mRNA stabilizedongoing TfR synthesisongoing TfR synthesiswhen IRP is bound to the IREwhen IRP is bound to the IRE binding of ribonuclease to binding of ribonuclease to rapid rapid turnover domain (RTD)turnover domain (RTD) is blocked is blocked

Ferric Ferric

reductasereductase

ferroportinferroportin(IReg1)(IReg1)

DMT1DMT1

hephaestinhephaestin

Fe Fe 2+2+


Fe Fe 2+2+

Fe Fe 3+3+

Fe Fe 2+2+

modulation of iron absorption by intestinal villi

Mature enterocytesMature enterocytes

Crypt cellCrypt cell


Fe Fe 3+3+

Fe Fe 2+2+

DMT1 mRNADMT1 mRNA

Crypt Cell Villus Enterocyte

Low iron state

Liver

Hepcidin TfR2

Holotransferrin

FeFe

+

Inflammatory Stimuli (IL-6;Lipopolysaccharide)

+

Fe2+Fe2+

Macrophage_

Fe

Fe

_

Villusenterocyte

DMT1

_

Ferric reductase

Fe+3 Fe+2

Erythropoietin

_

FPN1Ferritin

FPN1

Fe2+

Fe+2

CP

Fe+3

TNF-α

_

Hemojuvelin+

Hypoxia

Crypt cellFe2+

IRP IRP-Fe

3’

mRNA(DMT1)

5’

Proteins of iron transport• Transferrin / FerritinTransferrin / Ferritin• Transferrin receptors: TfR1, TfR2 • Ceruloplasmin / Hephaestin• Divalent Metal Transporter 1• Ferroportin • IRP1 and IRP2 (cytosolic mRNA binding)• HFE protein• ß-2 microglobulin• DCyt B (ferric reductase)• Hepcidin• Hemojuvelin

Types of Hemochromatosis

Type of HH Gene Protein Gene mapping

Type of inheritance

Classic hemochromatosis (HFE1) – later onset

HFE7 exons

HFE(non-classical MHC class-I protein)

6p21.3 Autosomal recessive

Juvenile hemochromatosis: HFE2A

HFE2B

HJV4 exons

HAMP(LEAP1)3 exons

Hemojuvelin(hemojuvelin precursor)

Hepcidinantimicrobial peptide

1q21

19q13

Autosomal recessive

Autosomal recessive

Hemochromatosis, type 3 (HFE3) – later onset

TfR218 exons

Transferrin receptor 2

7q22 Autosomal recessive

Hemochromatosis, type 4 (HFE4) (ferroportin disease)

FPN18 exons

Ferroportin1(iron-regulated transporter-1)

2q32 Autosomal dominant

Classical or adult-onset Hemochromatosis

Classical Hemochromatosis

• First description 1865

• inherited disorder 1935– autosomal recessive disorder of excess iron

deposits in parenchymal tissues causing organ damage and dysfunction

– Affects liver, pancreas, heart, joints, pituitary, skin – “bronze diabetes”

• Considered rare disease of elderly men

Hemochromatosis by 1996• Syndrome preventable if iron overload diagnosed

and treated early. – Treatment simple: - phlebotomy

• Recognition – high transferrin saturation and ferritin• Studies of blood donors suggested that 1:200 to

1:400 people have biochemical iron overload

• Much more common than originally thought– 1 in 200 in NW European populations

C282Y mutation in HFE geneC282Y mutation in HFE gene

B C E A B C E A HLA genesHLA genes

HFEHFE

In late 1996 an HLA linked gene on In late 1996 an HLA linked gene on chromosome 6 p 21.3 was found to be chromosome 6 p 21.3 was found to be

associated with hemochromatosis patients associated with hemochromatosis patients of of

North West European originNorth West European origin

HFE mutations in Caucasians

MMuuttaattiioonn NNuucclleeoottiiddee AAmmiinnooaacciidd

CC228822YY

GG AA 884455

CCyyss ttoo TTyyrr

HH6633DD

CC GG 118877

HHiiss ttoo AAsspp

A single mutation, A single mutation, C282YC282Y was shown to be was shown to be associated with hemochromatosis in around associated with hemochromatosis in around

80%80% of patients of of patients of NW EuropeanNW European origin origin

• Genetics: Homozygosity for C282Y very common in NW Europeans (1:200)

• Biochemistry: Most homozygotes will slowly accumulate iron leading to high ferritin and transferrin saturation > 55%

• Clinical: Disease penetrance very variable from early symptoms and severe disease to no symptoms – genetic diagnosis very common – but the disease syndrome much less so

• Study of 26,000 genotyped subjects from San Diego Kaiser Permanente clinic

• (Beutler: Lancet 2002;211-128)

• 152 homozygotes – only 1 with clinical syndrome – penetrance 1%

• Fatigue, arthralgias, impotence, arrhythmias no more prevalent than in non-C282Y homozygotes

• Only significant difference was 5-10% had abnormal liver function tests

Ferritin by gender: C282Y homozygotes

0

200

400

600

800

1000

1200

1400

1600

1800

2000

0 10 20 30 40Age (years)

ug/L

Female

Male

Transferrin Saturation* by gender: C282Y homozygotes

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 10 20 30 40Age (years)

Female

Male

* calculated

C282YC282Y

Family - HFE.1.

Adult-onset Hemochromatosis

due to TfR2(transferrin receptor 2)

mutations

Referred for HFE testing• 35 year old man

• severe iron overload– ferritin 34,000– saturation 0.90

• results: C282Y wt / wt H63D wt/ mut

TRF2 Study:TRF2 Study: Mattman, Vatcher, Ralston Mattman, Vatcher, RalstonHuntsman, Lockitch, Langlois et alHuntsman, Lockitch, Langlois et al

5’ 3’

Y250X

A376D N402K R752H

Q690P

X4X3

Homozygous

Heterozygous

E60X M172K

A75V I238M*

Previously described homozygous mutations

Novel homozygous mutation

Novel heterozygous sequence variation

* Previously described sequence variantMattman et al. Blood: 2002; 100; 1075-7

2

7

I

II

III

IV36 35 30 25

567 Ferritin

45 Sat

23477

3595

3400090

3930

1

10-11986 71-5

11103-921

2

4321

Q690P

Age

Ferritinsaturation

Patient

IV-2

III-8

IV-I

IV-3

IV-4

Genotype

ccg homozygotepro/pro

cag/ccg heterozygotegln/pro

cag homozygotegln/gln



Juvenile HemochromatosisType 2.A Hemojuvelin

Type 2.B Hepcidin

Juvenile Hemochromatosis

• autosomal recessive disorder• affects male and female equally• Usually presents between 10 and 30 years• severe iron overload, organ failure and high

mortality rate • hypogonadism and cardiomyopathy are

prominent features• Also cirrhosis, diabetes, arthopathy

Juvenile Hemochromatosis

• Of the first 16 reported cases – diagnosed at 4 – 19 years

– 11 died within 2 years of diagnosis• Congestive heart failure• Severe cirrhosis and liver failure

• A 7 year old girl saw her GP because her teacher thought she looked very pale.

• Her blood count parameters were normal but her ferritin was 339 and transferrin saturation 0.94

• Her younger siblings also had high ferritin and saturation

Family JH.1 1987

Age 7Age 7 Sat 0.94 Sat 0.94 ferritin 339ferritin 339



Sat 0.26Sat 0.26 ferritin 81ferritin 81

Sat Sat 0.330.33 ferritin 95ferritin 95

Family JH.1

Liver iron content

7 yr 6 yr 4 yr N

hepatic iron 8254 6582 4679 < 290

HII 21.1 19.6 20.9 1

2 years later after regular phlebotomy

hepatic iron 1588 795 2141

HII 3.16 1.58 7.67

ferritin 15 21 35

sat 0.6 0.8 0.9

Family JH.1




SatSat 0.260.26 ferritin 81ferritin 81

SatSat 0.330.33 ferritin 95ferritin 95

C282YC282Y

H63DH63D

1997 HFE testing

Family JH.1




Sat Sat 0.260.26 ferritin 81ferritin 81


I222NI222N

G320VG320V

HFE2 (HJV) hemojuvelinTesting

Family JH.1 2004

Eleven years post diagnosis

• Treated rigorously with phlebotomies ever since diagnosis

• Seen at 18, 17 and 14 years respectively

• No evidence of cardiac, hepatic or endocrine dysfunction

• A 19 year old boy presented in severe cardiomyopathic heart failure. He was a candidate for heart transplantation

• Transferrin saturation was 100% and ferritin 1403

• Following intensive phlebotomy therapy cardiac function improved dramatically and transplant was avoided.

Family JH.2

Age 19Age 19Sat 1.00 Sat 1.00 ferritin 1403ferritin 1403


Sat 0.26 Sat 0.26 ferritin 90ferritin 90



C282YC282YFamily JH.2

1997 HFE testing





2121 1919


UndefinedUndefined

G320VG320V

HFE2 (HJV) Testing

Family JH.2 2004

hemojuvelin

Mutations identified in hemojuvelin gene

G320V

R326X

C361fsX366

I222NG99VI281T

exon 1 exon 2 exon 3 exon 4

G320V Found in Greek, French, Irish, Scottish, American, Canadian, Australian, Croatian,

Slovakian, German patients so far

Juvenile Hemochromatosis: Type 2B

• Severe early onset – phenotypically indistinguishable from Type 2A

• No linkage to chromosome 1

• Two families with mutations in the hepcidin gene originally described from Italy

• Suggested hepcidin and hemojuvelin function together in iron signaling

Juvenile Hemochromatosis• presentation in 26 subjects

– mean age of 23.3 years – ferritin > 3000– Sat > 90%

Hypogonadism 96%

Cardiomyopathy 35%

Impaired glucose tolerance 60%

Cirrhosis 42%

Arthropathy 27%

Autosomal Dominant Hemochromatosis

Ferroportin Disease

• A 45 year old woman has a persistently high ferritin around 2250 and saturation of 0.65, necessitating ongoing phlebotomy

– Phlebotomy rapidly induces anemia

• Her 4 children have high ferritin levels but their transferrin saturations are normal

• ΔΔ HHCS: Hereditary Hyperferritinemia Cataract Syndrome

Family FD.1

ferritin 150 ferritin 150 Sat 0.25Sat 0.25


WTWT

C282YC282Y

Age 15Age 15ferritin 274ferritin 274Sat 0.35 Sat 0.35

Age 20Age 20ferritin 686ferritin 686Sat 0.24Sat 0.24


Age 17Age 17ferritin 442 ferritin 442 Sat 0.25 Sat 0.25

Family FD.1

1999 HFE1 Testing

2001

• Autosomal dominant hemochromatosis described in Italian and Dutch families

• Mutations found in the ferroportin gene

• Followed rapidly by reports from other parts of the world- not a rare disorder



WTWT

FPN N185DFPN N185D

Age 15Age 15ferritin 274ferritin 274Sat 0.35 Sat 0.35



Age 17Age 17ferritin 442 ferritin 442 Sat 0.25 Sat 0.25

Family FD.1 2005 Ferroportin gene testing

Novel mutation and polymorphism in FPN1 exon 6

N185D Mutation

Wild TypeGenotype

23-45 96-115 127-152 206-228 307-324 343-362 374-393 450-471 518-537

43bp 68bp 160bp 116bp 127bp 246bp 642bp 314bp

3’UTR

exon 1 exon 2 exon 3 exon 4 exon 5 exon 6 exon 7 exon 8

IRE

N144DN144HN144TA77D V162del

D157G

Q182H G323V

Y64N G80S

Q248H

D270V C326Y

G490D

N174I N185D

427bp 1,286bp

5’UTR

Gene:

Protein:

1-14 15-37 38-90 91-129 130-171 172-253 254-467 468-571

H2N COOH

V162del - Australia, Italy, UK, Greece

A77D - Italy, Australia

Common mutations

N185DNovel mutation

61-80 186-203 493-512

,

I

II

III

IV

V

VI

1 2 3 4 5 6 7

1 2 3 4 5 6 7 8 9 10 11

1 2 3 4

5 6 7 8

1 2 Heterozygous for FPN N185DHeterozygous for FPN N185D

Family 4.1

20 19 1517 6 6 2083 children

S age allele ferritin sat phlebotomy

V.1 20 F N185D 686 24 Ongoing

V.2 19 M N185D 977 46 Ongoing

V.3 17 M N185D 442 25 Ongoing

V.4 15 F N185D 274 35 Ongoing

V.5 8 F WT 35 0.26 n/a

V.6 6 M N185D 109 25 new dx

V.7 6 M N185D 220 25 new dx

V.8 15 M N185D 271 18 new dx

Autosomal Dominant Hemochromatosis (HFE4)

• Mutations in ferroportin gene

• Iron loading initially in RE cells Transferrin saturation moderate despite high ferritin Ferroportin export of iron from macrophages reduced

• In some cases tend to develop anemia quickly on phlebotomy

Autosomal Dominant Hemochromatosis (HFE4)

• Suspect in patient with persistent high ferritin, not otherwise explained and low or normal saturation

• Suspect in families with apparent autosomal dominant hemochromatosis – caveat - HFE1

• If ferroportin mutation found even young children should have molecular testing

• If no mutation can rule out need for further iron monitoring

Points to ponder

Points to Ponder

• The difference between genotype and clinical disease– examples

• How early should children be tested?– for C282Y– for ferroportin mutations

What accounts for the difference between genotype and clinical

disease?

• A 17 year old high school student presents with a one year history of intractable fatigue

• He has been seen by various specialists including internal medicine and rheumatology

• His paternal aunt has just been diagnosed with hemochromatosis

Family HFE:2





C282YC282Y

WTWT

HFE testHFE test

Family HFE:2

HFE by report

Age 17Age 17 Age 15Age 15

• A 5 year old girl presents with vague history of recurrent abdominal pain

• She has a transferrin saturation of 0.85 and ferritin of 48

• Her mother had gall-bladder surgery at 21 and was found to have iron overload - NYD

Family HFE:3

5 years 5 years Sat 0.85 Sat 0.85 ferritin 46ferritin 46

9 years9 yearsSat 0.27 Sat 0.27 ferritin 32ferritin 32

0.620.62600600

C282YC282Y

H63DH63D

Diagnosed at 21 with iron overload and high liver ironPhlebotomized since

Sat 0.34Sat 0.34ferritin 573ferritin 573

Family HFE:3





2121 1919


UndefinedUndefined

G320VG320V

HFE2 (HJV) Testing

Family JH.2 2004

hemojuvelin

Points to ponder

• Age 21

• Sat 0.98

• ferritin 2467

• No evidence of cardiomyopathy


CardiomyopathyCardiomyopathyHypogonadismHypogonadismplusplusBannayan-Riley-Bannayan-Riley-Ruvalcaba syndromeRuvalcaba syndrome(macrocephaly, hemangiomas, (macrocephaly, hemangiomas, lipomas)lipomas)

How early should children be tested?

for C282Y?

for ferroportin mutations?

Ferritin by gender: C282Y homozygotes

0

200

400

600

800

1000

1200

1400

1600

1800

2000

0 10 20 30 40Age (years)

ug/L

Female

Male

Transferrin Saturation* by gender: C282Y homozygotes

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 10 20 30 40Age (years)

Female

Male

* calculated

S age allele ferritin sat phlebotomy

V.1 20 F N185D 686 24 Ongoing

V.2 19 M N185D 977 46 Ongoing

V.3 17 M N185D 442 25 Ongoing

V.4 15 F N185D 274 35 Ongoing

V.5 8 F WT 35 0.26 n/a

V.6 6 M N185D 109 25 new dx

V.7 6 M N185D 220 25 new dx

V.8 15 M N185D 271 18 new dx

Guidelines

The Clinical Laboratory in BC

• Apply to classical Type 1 Hemochromatosis

• General guidelines – indications for genetic testing

• Based on fasting transferrin saturation as the primary biochemical screen

BC Guidelines: Iron Overload 2001

Indications to consider hemochromatosis

When to consider the diagnosis?

• Adult onset diabetes• Arthritis• Unexplained cirrhosis or persistently raised liver enzymes•Congestive heart failure or cardiomyopathy•Secondary hypogonadism•Increased skin pigmentation

• Not in guideline• Severe fatigue• Arthralgias

The Clinical Laboratory in BC

• Ferritin and transferrin saturation (fTS)

• Indication for genetic test for C282Y mutation– fTS ≤ 0.45 not indicated – fTS 0.45 to 0.60 may repeat in a month– fTS ≥ 60 suggest genetic test2001 guidelinesdepends on clinical picture

Test done in Children’s Hospital Molecular Diagnostic Lab and other referral labs.

Genetic Testing and Treatment

• First degree relatives of confirmed hemochromatosis patients can have the genetic test done directly

• If iron overloaded and not C282Y homozygous consider other causes

• Management (phlebotomy) is dependent on the ferritin level not the transferrin saturation

• Phlebotomy - till ferritin around 50 µg/L

Take home messages

Take Home Messages

Hemochromatosis is not an “old man’s disease”Biochemical iron overload occurs in young adults

The most severe form of hemochromatosis – Juvenile Hemochromatosis -occurs in children and young adults – though rare

There are now at least 4 other genes than HFE1 in which mutations cause hemochromatosis

Take Home Messages

If suspected: measure transferrin saturation and ferritin.

Elevated saturation – genetic testing

High ferritin – consider phlebotomy

Classical hemochromatosis:

Molecular hemochromatosis (C282Y homozygosity) 1:200

but clinical disease much less frequent

Take Home Messages

Suspect:

Unexplained cardiomyopathy

Hypogonadism: delayed puberty

Look for very high transferrin saturation and hyperferritinemia

Juvenile Hemochromatosis:

Rare but much more lethal if diagnosis missed

Take Home Messages

If there is an apparent autosomal dominant pattern of hemochromatosis think of ferroportin disease

but remember prevalence of classical hemochromatosis can result in pseudo-dominant pattern

Acknowledgements Diana Ralston Paul Goldberg

Tara Morris Julie MacFarlane

Mariya Litvinova Patrice Eydoux Andre Mattman Patrick MacLeod

Dan Holmes Sylvie Langlois

Yigal Kaikov

Special Funding from the President’s Award,

Russian Academy of Science (Mariya Litvinova)

hemochromatosis bcsls telehealth presentation october 19, 2006 gillian lockitch, mbchb, md, frcpc

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