Hemochromatosis

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Hemochromatosis

• BCSLS Telehealth Presentation
• October 19, 2006
• Gillian Lockitch, MBChB, MD, FRCPC

2
OBJECTIVES

• Review iron absorption and transport
• Describe types of hemochromatosis
• Hemochromatosis and the laboratory
• Suspicion
• Investigation
• Diagnosis

3
Iron Absorption and Transport
4
Fe
Fe
Villus enterocyte
Fe2
Fe3
Fe2
Ferritin
Macrophage
Ceruloplasmin
Bone Marrow
Liver
5
Normal Iron metabolism
Male
Female

• 3 - 4 g
• 1 2 mg
• 1- 2 mg

• Total body iron
• Daily iron absorption
• Daily iron loss
• OTHER
• Menstrual (monthly)
• pregnancy (total)

• 2 - 3 g
• 1 2 mg
• 1- 2 mg
• OTHER
• 20 - 40 mg
• 600 - 900 mg

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Macrophage
Fe
Fe
Fe2
Villus enterocyte
20 mg / d
Liver
Menstruation 20 40 mg /m
Bone Marrow
7
Macrophage
Fe
Fe
Fe2
Villus enterocyte
20 mg / d
Liver
Menstruation 20 40 mg /m
Bone Marrow
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Iron transport and absorption proteins

• Transferrin
• Ferritin
• Transferrin receptor
• Ceruloplasmin

9
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

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Fe 3
Fe 2
Reduction of ferric to ferrous iron
Ferric reductase
Transport across brush border
Divalent metal transporter 1 (DMT1)
Ferric reductase
DMT1
Sequestration in enterocyte
Ferritin
Basal transport from cell
Ferroportin
Hephaestin
Ferroportin (IReg1)
Oxidation to ferric form
Hephaestin Ceruloplasmin
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Iron regulates the synthesis of its own
key transport and storage proteins
Iron responsive elements (IREs) Iron responsive
element binding proteins (IRPs)
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(No Transcript)
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Iron Responsive Elements (IREs)
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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)

15
Ferritin
5
16
DMT1
Control of synthesis
IREs
Low intracellular iron content
mRNA transcript
3
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
17
Fe 3
Fe 2
Ferric reductase
DMT1
hephaestin
ferroportin (IReg1)
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modulation of iron absorption by intestinal villi
Mature enterocytes
Crypt cell
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Villus Enterocyte
Crypt Cell
Fe 3
Fe 2
Low iron state
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Fe3
Fe2
DMT1
Fe
Ferric reductase
Fe
Fe2
Fe2
FPN1
Ferritin
Villus enterocyte
Hypoxia
_
_
Erythropoietin
Macrophage
_
mRNA(DMT1)
3
5
Hemojuvelin

IRP
IRP-Fe
Crypt cell
Hepcidin
TfR2
FPN1
Fe2

_
_
Liver
TNF-a
Fe
Fe
Holotransferrin

Inflammatory Stimuli (IL-6 Lipopolysaccharide)
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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

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Types of Hemochromatosis
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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
Juvenile hemochromatosis HFE2A HFE2B HJV 4 exons HAMP (LEAP1) 3 exons Hemojuvelin (hemojuvelin precursor) Hepcidin antimicrobial peptide 1q21 19q13 Autosomal recessive 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
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Classical or adult-onset Hemochromatosis
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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

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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 1200 to
  1400 people have biochemical iron overload
• Much more common than originally thought
• 1 in 200 in NW European populations

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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
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HFE mutations in Caucasians
A single mutation, C282Y was shown to be
associated with hemochromatosis in around 80 of
patients of NW European origin
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• Genetics Homozygosity for C282Y very common in
  NW Europeans (1200)
• Biochemistry Most homozygotes will slowly
  accumulate iron leading to high ferritin and
  transferrin saturation gt 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

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• Study of 26,000 genotyped subjects from San Diego
  Kaiser Permanente clinic
• (Beutler Lancet 2002211-128)
• 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

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calculated
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Family - HFE.1.
C282Y
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Adult-onset Hemochromatosisdue to
TfR2(transferrin receptor 2)mutations
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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 Mattman, Vatcher, Ralston Huntsman,
Lockitch, Langlois et al
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Y250X
Q690P
E60X
M172K
Homozygous
5
3
Heterozygous
A376D
N402K
R752H
A75V
I238M
X4
X3
Previously described homozygous mutations
Novel homozygous mutation
Novel heterozygous sequence variation
Mattman et al. Blood 2002 100 1075-7

Previously described sequence variant
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1
2
I
11
10
3-9
2
1
II
7
10-11
9
8
7
6
1-5
III
5
2
67 Ferritin 45 Sat
3
2
4
1
IV
36
35
30
25
Age
Ferritin saturation
35 95
39 30
234 77
34000 90
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Patient
Genotype
ccg homozygote pro/pro cag/ccg
heterozygote gln/pro cag homozygote gln/gln ccg
homozygote pro/pro ccg homozygote pro/pro
IV-2 III-8 IV-I IV-3 IV-4
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Juvenile Hemochromatosis

• Type 2.A Hemojuvelin
• Type 2.B Hepcidin

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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

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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

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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

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Family JH.1
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Liver iron content
Family JH.1

• 7 yr 6 yr 4 yr N
• hepatic iron 8254 6582 4679 lt 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

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Family JH.1
1997 HFE testing
C282Y
H63D
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Family JH.1 2004
HFE2 (HJV) hemojuvelin Testing
I222N
G320V
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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

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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.

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Family JH.2
C282Y
1997 HFE testing
Sat 0.21 ferritin 26
Sat 0.26 ferritin 90
Age 19 Sat 1.00 ferritin 1403
Age 19 Sat 0.24 ferritin 30
Age 21 Sat 0.98 ferritin 2467
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2004
Family JH.2
HFE2 (HJV) Testing
hemojuvelin
G320V
Sat 0.26 ferritin 90
Sat 0.21 ferritin 26
Undefined
21
19
Age 19 Sat 0.24 ferritin 30
Age 19 Sat 1.00 ferritin 1403
Age 21 Sat 0.98 ferritin 2467
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(No Transcript)
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Mutations identified in hemojuvelin gene
C361fsX366
I281T
R326X
G99V
I222N
G320V
exon 2
exon 3
exon 4
exon 1
G320V
Found in Greek, French, Irish, Scottish,
American, Canadian, Australian,
Croatian, Slovakian, German patients so far
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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

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Juvenile Hemochromatosis

• presentation in 26 subjects
• mean age of 23.3 years
• ferritin gt 3000
• Sat gt 90
• Hypogonadism 96
• Cardiomyopathy 35
• Impaired glucose tolerance 60
• Cirrhosis 42
• Arthropathy 27

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Autosomal Dominant Hemochromatosis

• Ferroportin Disease

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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
• ?? HHCS Hereditary Hyperferritinemia Cataract
  Syndrome

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Family FD.1
1999 HFE1 Testing
C282Y
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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

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2005 Ferroportin gene testing
Family FD.1
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Novel mutation and polymorphism in FPN1 exon 6
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N144D N144H N144T
D157G
Y64N
G80S
D270V
C326Y
N174I
N185D
Gene
A77D
V162del
G490D
3UTR
Q182H
G323V
Q248H
5UTR
427bp
1,286bp
43bp
68bp
160bp
116bp
127bp
246bp
642bp
314bp
IRE
exon 1
exon 2
exon 3
exon 4
exon 5
exon 6
exon 7
exon 8
1-14
15-37
38-90
91-129
130-171
172-253
254-467
468-571
23-45
96-115
127-152
206-228
307-324
343-362
374-393
450-471
518-537
61-80
186-203
493-512
H2N
COOH
Protein
Novel mutation
N185D
Common mutations
,
V162del - Australia, Italy, UK, Greece
A77D - Italy, Australia
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Family 4.1
I
II
III
1 2 3 4
5 6 7
IV
1
2 3 4
5 6 7
8 9 10 11
V
20
19
15
17
6
6
20
8
3 children
1 2 3 4
5 6 7 8
VI
Heterozygous for FPN N185D
1 2
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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


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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

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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

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Points to ponder
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Points to Ponder

• The difference between genotype and clinical
  disease
• examples
• How early should children be tested?
• for C282Y
• for ferroportin mutations

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What accounts for the difference between genotype
and clinical disease?
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Family HFE2

• 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

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Family HFE2
Sat 0.21 ferritin 26
Sat 0.93 ferritin 641
Sat 0.25 ferritin 33
HFE by report
Age 15
Age 17
Sat 0.90 ferritin 560
71
Family HFE3

• 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

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Family HFE3
0.62 600
Sat 0.34 ferritin 573
Diagnosed at 21 with iron overload and high liver
iron Phlebotomized since
5 years Sat 0.85 ferritin 46
C282Y
9 years Sat 0.27 ferritin 32
H63D
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2004
Family JH.2
HFE2 (HJV) Testing
hemojuvelin
G320V
Sat 0.26 ferritin 90
Sat 0.21 ferritin 26
Undefined
21
19
Age 19 Sat 0.24 ferritin 30
Age 19 Sat 1.00 ferritin 1403
Age 21 Sat 0.98 ferritin 2467
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Points to ponder

• Age 21
• Sat 0.98
• ferritin 2467
• No evidence of cardiomyopathy

Age 19 Sat 1.00 ferritin 1403 Cardiomyopathy Hy
pogonadism plus Bannayan-Riley-Ruvalcaba
syndrome (macrocephaly, hemangiomas, lipomas)
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How early should children be tested?

• for C282Y?
• for ferroportin mutations?

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(No Transcript)
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calculated
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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


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Guidelines
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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

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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

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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 Childrens Hospital Molecular
  Diagnostic Lab and other referral labs.

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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

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Take home messages
85
Take Home Messages
Hemochromatosis is not an old mans
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
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Take Home Messages
Classical hemochromatosis Molecular
hemochromatosis (C282Y homozygosity) 1200 but
clinical disease much less frequent
If suspected measure transferrin saturation and
ferritin. Elevated saturation genetic
testing High ferritin consider phlebotomy
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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
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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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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 Presidents Award,
• Russian Academy of Science (Mariya Litvinova)