hemochromatosis bcsls telehealth presentation october 19, 2006 gillian lockitch, mbchb, md, frcpc
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OBJECTIVES
• Review iron absorption and transport
• Describe types of hemochromatosis
• Hemochromatosis and the laboratory – Suspicion– Investigation – Diagnosis
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
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+
Fe Fe 3+3+
Fe Fe 2+2+
modulation of iron absorption by intestinal villi
Mature enterocytesMature enterocytes
Crypt cellCrypt cell
Fe Fe 2+2+Fe Fe 3+3+
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
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 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
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
ccg homozygotepro/pro
ccg homozygotepro/pro
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
Age 6Age 6 Sat 0.90 Sat 0.90 ferritin 146ferritin 146
Age 4Age 4 Sat 0.59 Sat 0.59 ferritin 187ferritin 187
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
Age 7Age 7 Sat 0.94 Sat 0.94 ferritin 339ferritin 339
Age 6Age 6 Sat 0.90 Sat 0.90 ferritin 146ferritin 146
Age 4Age 4 Sat 0.59 Sat 0.59 ferritin 187ferritin 187
SatSat 0.260.26 ferritin 81ferritin 81
SatSat 0.330.33 ferritin 95ferritin 95
C282YC282Y
H63DH63D
1997 HFE testing
Family JH.1
Age 7Age 7 Sat 0.94 Sat 0.94 ferritin 339ferritin 339
Age 6Age 6 Sat 0.90 Sat 0.90 ferritin 146ferritin 146
Age 4Age 4 Sat 0.59 Sat 0.59 ferritin 187ferritin 187
Sat Sat 0.260.26 ferritin 81ferritin 81
Sat 0.33Sat 0.33 ferritin 95ferritin 95
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
Age 21Age 21Sat 0.98 Sat 0.98 ferritin 2467ferritin 2467
Sat 0.26 Sat 0.26 ferritin 90ferritin 90
Sat 0.21 Sat 0.21 ferritin 26ferritin 26
Age 19Age 19Sat 0.24 Sat 0.24 ferritin 30ferritin 30
C282YC282YFamily JH.2
1997 HFE testing
Age 19Age 19Sat 1.00 Sat 1.00 ferritin 1403ferritin 1403
Age 21Age 21Sat 0.98 Sat 0.98 ferritin 2467ferritin 2467
Sat 0.26 Sat 0.26 ferritin 90ferritin 90
Sat 0.21 Sat 0.21 ferritin 26ferritin 26
2121 1919
Age 19Age 19Sat 0.24 Sat 0.24 ferritin 30ferritin 30
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%
• 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
ferritin 2248 ferritin 2248 Sat 0.65Sat 0.65
WTWT
C282YC282Y
Age 15Age 15ferritin 274ferritin 274Sat 0.35 Sat 0.35
Age 20Age 20ferritin 686ferritin 686Sat 0.24Sat 0.24
Age 19Age 19ferritin 977ferritin 977Sat 0.48Sat 0.48
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
ferritin 150 ferritin 150 Sat 0.25Sat 0.25
ferritin 2248 ferritin 2248 Sat 0.65Sat 0.65
WTWT
FPN N185DFPN N185D
Age 15Age 15ferritin 274ferritin 274Sat 0.35 Sat 0.35
Age 20Age 20ferritin 686ferritin 686Sat 0.24Sat 0.24
Age 19Age 19ferritin 977ferritin 977Sat 0.48Sat 0.48
Age 17Age 17ferritin 442 ferritin 442 Sat 0.25 Sat 0.25
Family FD.1 2005 Ferroportin gene testing
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
• The difference between genotype and clinical disease– examples
• How early should children be tested?– for C282Y– for ferroportin mutations
• 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
Sat 0.25Sat 0.25 ferritin 33ferritin 33
Sat 0.90 Sat 0.90 ferritin 560ferritin 560
Sat 0.93 Sat 0.93 ferritin 641ferritin 641
Sat 0.21Sat 0.21 ferritin 26ferritin 26
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
Age 19Age 19Sat 1.00 Sat 1.00 ferritin 1403ferritin 1403
Age 21Age 21Sat 0.98 Sat 0.98 ferritin 2467ferritin 2467
Sat 0.26 Sat 0.26 ferritin 90ferritin 90
Sat 0.21 Sat 0.21 ferritin 26ferritin 26
2121 1919
Age 19Age 19Sat 0.24 Sat 0.24 ferritin 30ferritin 30
UndefinedUndefined
G320VG320V
HFE2 (HJV) Testing
Family JH.2 2004
hemojuvelin
Points to ponder
• Age 21
• Sat 0.98
• ferritin 2467
• No evidence of cardiomyopathy
Age 19Age 19Sat 1.00 Sat 1.00 ferritin 1403ferritin 1403
CardiomyopathyCardiomyopathyHypogonadismHypogonadismplusplusBannayan-Riley-Bannayan-Riley-Ruvalcaba syndromeRuvalcaba syndrome(macrocephaly, hemangiomas, (macrocephaly, hemangiomas, lipomas)lipomas)
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
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
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
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