Hemochromatosis
BCSLS Telehealth Presentation
October 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
Fe+3
Fe+2
Fe
Villus
enterocyte
Fe
Fe2+
Fe+2
Ceruloplasmin
Fe+3
Fe+3
Fe
Ferritin
Macrophage
Fe
Transferrin
Fe+2
Bone Marrow
Liver
Normal Iron metabolism
Male
• 3-4g
Female
• 2-3g
• Daily iron
absorption
• 1 – 2 mg
• 1 – 2 mg
• Daily iron loss
• 1- 2 mg
•
• Total body iron
OTHER
• Menstrual (monthly)
• pregnancy (total)
1- 2 mg
OTHER
• 20 - 40 mg
• 600 - 900 mg
Fe+3
Fe+2
Ferritin
Fe
Fe
Macrophage
Fe2+
Villus
enterocyte
1 – 2 mg /d
20 mg / d
1 – 2 mg /d
Liver
Menstruation
20 – 40 mg /m
Bone Marrow
Fe+3
Fe+2
Ferritin
Fe
Fe
Macrophage
Fe2+
Villus
enterocyte
20 mg / d
7 – 8 mg /d
1 – 2 mg /d
Liver
Menstruation
20 – 40 mg /m
Bone Marrow
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
Fe 3+
Fe 2+
Reduction of ferric to ferrous iron
Ferric reductase
Transport across brush border
Ferric
reductase
Fe
2+
DMT1
Divalent metal transporter 1
(DMT1)
Sequestration in enterocyte
Ferritin
Fe 2+
Basal transport from cell
Ferroportin
Ferroportin
(IReg1)
Hephaestin
Oxidation to ferric form
Hephaestin
Ceruloplasmin
Iron regulates the synthesis of its
own key
transport and storage proteins
Iron responsive elements (IREs)
Iron responsive element binding
proteins (IRPs)
DNA
transcription
Messenger RNA
translation
Protein
Iron Responsive Elements (IREs)
Low intracellular iron:
IRP binds and prevents
mRNA translation
IRE
Low intracellular iron:
IRP binds and stabilizes
mRNA
mRNA transcript
5`
IRE
3`
Untranslated regions
IRP1 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)
Ferritin
Low intracellular iron content
IRP bound
mRNA not translated
ferritin low
mRNA transcript
5`
High intracellular iron content
IRP not bound
mRNA translated
ferritin synthesized
DMT1
Control of synthesis
Low intracellular iron content
IREs
mRNA transcript
RTD
IRPs bound - mRNA stabilized
ongoing TfR synthesis
when IRP is bound to the IRE
binding of ribonuclease to rapid
turnover domain (RTD) is blocked
3`
Fe 3+
Ferric
reductase
Fe 2+
Fe 2+
DMT1
Fe 2+
ferroportin
(IReg1)
hephaestin
modulation of iron absorption
by intestinal villi
Mature enterocytes
Crypt cell
Villus Enterocyte
Crypt Cell
Fe 3+
DMT1 mRNA
Low iron state
Fe 2+
Fe+3
Fe+2
DMT1
Fe
Ferric
reductase
Fe+3
Fe
CP
Fe2+
Fe+2
FPN1
Villus
enterocyte
Fe2+
Ferritin
Hypoxia
_
_
Erythropoietin
Macrophage
_
mRNA(DMT1)
5’
3’
+
IRP
Hemojuvelin
IRP-Fe
Crypt cell
Fe2+
Hepcidin
FPN1
_
Liver
+
_
TNF-α
+
Inflammatory
Stimuli (IL-6;
Lipopolysaccharide)
TfR2
Fe
Fe
Holotransferrin
Proteins of iron transport
•
•
•
•
•
•
•
•
•
•
•
Transferrin / 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
HFE
7 exons
HFE
(non-classical
MHC class-I
protein)
6p21.3
Autosomal
recessive
HJV
4 exons
Hemojuvelin
(hemojuvelin
precursor)
1q21
Autosomal
recessive
HFE2B
HAMP
(LEAP1)
3 exons
Hepcidin
antimicrobial
peptide
19q13
Autosomal
recessive
Hemochromatosis,
type 3 (HFE3) – later onset
TfR2
18 exons
Transferrin
receptor 2
7q22
Autosomal
recessive
Hemochromatosis,
type 4 (HFE4)
(ferroportin disease)
FPN1
8 exons
Ferroportin1
(iron-regulated
transporter-1)
2q32
Autosomal
dominant
Juvenile hemochromatosis:
HFE2A
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 gene
In late 1996 an HLA linked gene on
chromosome 6 p 21.3 was found to be
associated with hemochromatosis patients
of
North West European origin
HFE
B
C E A
HLA genes
HFE mutations in Caucasians
A single mutation, C282Y was shown to be
associated with hemochromatosis in around
80% of patients of NW European origin
Mutation
Nucleotide Aminoacid
C 28 2Y
GA
8 45
Cys to Tyr
H 63 D
CG
1 87
His to Asp
• 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
Female
Male
ug/L
2000
1800
1600
1400
1200
1000
800
600
400
200
0
0
10
20
Age (years)
30
40
Transferrin Saturation* by gender:
C282Y homozygotes
Female
Male
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
* calculated
10
20
Age (years)
30
40
Family - HFE.1.
C282Y
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
H63D
wt / wt
wt/ mut
TRF2 Study: Mattman, Vatcher, Ralston
Huntsman, Lockitch, Langlois et al
E60X
M172K Y250X
Q690P
Homozygous
5’
3’
Heterozygous
A75V
I238M* A376D N402K
R752H
X3
X4
Previously described homozygous mutations
Novel homozygous mutation
Novel heterozygous sequence variation
*
Previously described sequence variant
Mattman et al. Blood:
2002; 100; 1075-7
1
I
II
III
1
2
2
3-9
10
11
7
1-5
5
7
6
8
9
10-11
2
67 Ferritin
45 Sat
2
1
IV
Age
36
Ferritin
39
saturation 30
3
4
35
30
25
34000
90
35
95
234
77
Q690P
Patient
Genotype
IV-2
ccg homozygote
pro/pro
III-8
cag/ccg heterozygote
gln/pro
IV-I
cag homozygote
gln/gln
IV-3
ccg homozygote
pro/pro
IV-4
ccg homozygote
pro/pro
Juvenile Hemochromatosis
Type 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
Family JH.1
1987
• 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
Sat 0.33
ferritin 95
Age 7
Sat 0.94
ferritin 339
Sat 0.26
ferritin 81
Age 6
Sat 0.90
ferritin 146
Age 4
Sat 0.59
ferritin 187
Family JH.1
hepatic iron
HII
Liver iron content
7 yr
8254
21.1
6 yr
6582
19.6
4 yr
4679
20.9
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
N
< 290
1
Family JH.1
1997 HFE testing
Sat 0.33
ferritin 95
Sat 0.26
ferritin 81
C282Y
H63D
Age 7
Sat 0.94
ferritin 339
Age 6
Sat 0.90
ferritin 146
Age 4
Sat 0.59
ferritin 187
Family JH.1
Sat 0.33
ferritin 95
Sat 0.26
ferritin 81
HFE2 (HJV)
hemojuvelin
Testing
I222N
G320V
Age 7
Sat 0.94
ferritin 339
Age 6
Sat 0.90
ferritin 146
2004
Age 4
Sat 0.59
ferritin 187
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
Family JH.2
• 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
C282Y
1997 HFE testing
Sat 0.26
ferritin 90
Age 21
Sat 0.98
ferritin 2467
Sat 0.21
ferritin 26
Age 19
Sat 1.00
ferritin 1403
Age 19
Sat 0.24
ferritin 30
2004
Family JH.2
HFE2 (HJV) Testing
hemojuvelin
Sat 0.26
ferritin 90
21
Age 21
Sat 0.98
ferritin 2467
G320V
Undefined
Sat 0.21
ferritin 26
19
Age 19
Sat 1.00
ferritin 1403
Age 19
Sat 0.24
ferritin 30
Mutations identified in hemojuvelin gene
C361fsX366
I281T
G99V
exon 1
G320V
exon 2
I222N
exon 3
R326X
G320V
exon 4
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
Cardiomyopathy
Impaired glucose tolerance
Cirrhosis
Arthropathy
96%
35%
60%
42%
27%
Autosomal Dominant
Hemochromatosis
Ferroportin Disease
Family FD.1
• 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
1999 HFE1 Testing
WT
C282Y
ferritin 150
Sat 0.25
Age 20
ferritin 686
Sat 0.24
Age 19
ferritin 977
Sat 0.48
ferritin 2248
Sat 0.65
Age 17
ferritin 442
Sat 0.25
Age 15
ferritin 274
Sat 0.35
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
Family FD.1 2005 Ferroportin gene testing
WT
FPN N185D
ferritin 150
Sat 0.25
Age 20
ferritin 686
Sat 0.24
Age 19
ferritin 977
Sat 0.48
ferritin 2248
Sat 0.65
Age 17
ferritin 442
Sat 0.25
Age 15
ferritin 274
Sat 0.35
Novel mutation and polymorphism in
FPN1 exon 6
Wild Type
Genotype
N185D
Mutation
Y64N
G80S
A77D
Gene:
5’UTR
N144D D157G N174I N185D D270V C326Y
N144H
N144T V162del
Q182H Q248H
G323V
G490D
427bp
3’UTR
1,286bp
IRE 43bp
exon 1
1-14
68bp
160bp
exon 2
15-37
23-45
exon 3
38-90
61-80
96-115
91-129
127-152
116bp
127bp
exon 4
246bp
exon 5
130-171
186-203
exon 6
172-253
206-228
642bp
314bp
exon 7
exon 8
254-467
307-324
343-362
H2N
374-393
468-571
450-471
493-512
518-537
COOH
Protein:
Novel mutation
N185D
Common mutations
,
V162del - Australia,
Italy, UK, Greece
A77D
- Italy, Australia
Family 4.1
I
II
III
1
2
3
4
5
6
7
IV
1
V
2
20
19
1
2
3
4
17
15
3
4
5
6
7
8
9
10
8
3 children
5
11
6
6
20
6
7
8
VI
1
2
Heterozygous for FPN N185D
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
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
WT
35
0.26 n/a
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?
Family HFE:2
• 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 0.93
ferritin 641
Sat 0.21
ferritin 26
HFE test
WT
C282Y
Age 17
Sat 0.90
ferritin 560
Age 15
Sat 0.25
ferritin 33
HFE by
report
Family HFE:3
•
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
Sat 0.34
0.62
ferritin 573 600
C282Y
H63D
9 years
Sat 0.27
ferritin 32
Diagnosed at 21 with
iron overload and
high liver iron
Phlebotomized since
5 years
Sat 0.85
ferritin 46
2004
Family JH.2
HFE2 (HJV) Testing
hemojuvelin
Sat 0.26
ferritin 90
21
Age 21
Sat 0.98
ferritin 2467
G320V
Undefined
Sat 0.21
ferritin 26
19
Age 19
Sat 1.00
ferritin 1403
Age 19
Sat 0.24
ferritin 30
Points to ponder
• Age 21
• Sat 0.98
• ferritin 2467
• No evidence of
cardiomyopathy
Age 19
Sat 1.00
ferritin 1403
Cardiomyopathy
Hypogonadism
plus
Bannayan-RileyRuvalcaba syndrome
(macrocephaly, hemangiomas,
lipomas)
How early should children be
tested?
for C282Y?
for ferroportin mutations?
Ferritin by gender: C282Y homozygotes
Female
Male
ug/L
2000
1800
1600
1400
1200
1000
800
600
400
200
0
0
10
20
Age (years)
30
40
Transferrin Saturation* by gender:
C282Y homozygotes
Female
Male
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
* calculated
10
20
Age (years)
30
40
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
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
WT
35
0.26 n/a
Guidelines
The Clinical Laboratory in BC
Indications to consider hemochromatosis
BC Guidelines: Iron Overload 2001
• Apply to classical Type 1 Hemochromatosis
• General guidelines – indications for genetic testing
• Based on fasting transferrin saturation as the
primary biochemical screen
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 test
2001 guidelines
depends 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
There are now at least 4 other genes than HFE1
in which mutations cause hemochromatosis
The most severe form of hemochromatosis
– Juvenile Hemochromatosis -occurs in
children and young adults – though rare
Take Home Messages
Classical hemochromatosis:
Molecular hemochromatosis (C282Y
homozygosity) 1:200
but clinical disease much less frequent
If suspected: measure transferrin saturation
and ferritin.
Elevated saturation – genetic testing
High ferritin – consider phlebotomy
Take Home Messages
Juvenile Hemochromatosis:
Rare but much more lethal if diagnosis missed
Suspect:
Unexplained cardiomyopathy
Hypogonadism: delayed puberty
Look for very high transferrin saturation
and hyperferritinemia
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 pseudodominant pattern
Acknowledgements
Diana Ralston
Tara Morris
Mariya Litvinova
Andre Mattman
Dan Holmes
Yigal Kaikov
Paul Goldberg
Julie MacFarlane
Patrice Eydoux
Patrick MacLeod
Sylvie Langlois
Special Funding from the President’s Award,
Russian Academy of Science (Mariya Litvinova)