Transcript Slide 1

Hemophilia
Dr. Pupak Derakhshandeh (PhD)
Assis. Prof. Med. Sci. of Tehran Univ
.
Major bleeding disorders
(50%)
Hemophilia A (factor VIIIdeficiency)
Hemophilia B (factor IXdeficiency)
Clinically:
indistinguishable!
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Other bleeding disorders
(50%)
Factor XI- deficiency
Von Willebrand disease (VWB)
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Hemophilia (A/B)
phenotypic features
Severe bleeding and Hemorrhages
in the wounds, Joints, ankles,
knees, hips & elbows, muscles
Hematomas & Bruising
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Hemophilia A
The enlarged knee join
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Hemophilia A
The enlarged fore head
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Hemophilia A
Extensive bruising of the fore
arm and hand
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Hemophilia A/B
Severe:
Level of factor VIII/IX <1%
Moderate:
1% < Level of factor VIII/IX<5%
Mild:
5% < Level of factor VIII/IX <25%
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X-Linked Inheritance
Located on the X-Chromosome
More commonly affected males
Heterozygote female will pass the gene to
50% of her sons who will be affected,
and to 50% of her daughters who will be
carriers for the trait
Affected males pass the gene to all of
their daughters and none of their sons
Absence of male to male transmission
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How is ’Hemophilia’ inherited?
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X-Linked Inheritance
A pedigree of Hemophilia in European royal families
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Disease Etiology
Hemophilia A/B
Mutation in VIII & IX genus (a key
component of the clotting cascade)
Incidence:
Hemph.A1:5000-10.000 male
Hemph.B1: 30.000-100.000 male
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Mild Hemophilia in female
• Disadvantageous x-inactivation
• Rare: Severe disease
– Extreme skewing of X-inactivation
– Girl with Turner Syndrome
– Father: hemophiliac + Mother: Carrier
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Bleeding in Hemophilia
• Primary haemostasis: Normal
– Formation of a platelet plug
• Secondary haemostasis: Bleeding
– Stabilization of the plug by fibrin:
• Defective (inadequate amounts of
thrombin)
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Trifluorophosphine
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Genetics
Variability and Penetrance:
Fully penetrant
PND:
Just for severe Hemophilia
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Genetics
Hemophilia A
X-Linked recessive
Locus Xq 28
26 Exons / 186 kb DNA / 9 kb mRNA /
2,332 AA
Molecular Pathology:
Over 150 Mutations
45%:Inversion in intron 22
Other mutations (55%):
90-95% Point mutation
6% deletions
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Inhibitors
5-10 % of patient:
Don’t have a detectable mutation on
sequencing of Factor VIII
No native Factor VIII
develop anti-factor VIII
Antibodies with therapy
Complicate treatment!
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Antibodies that inactivate
Factor VIII in response to
treatment
• Hemophilia A:
– Incidence: 35 %
• Inversion in intron 22
• Large deletions
• Nonsense mutations
– Incidence: 5 %
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• Small deletion
• Missense mutations
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Genetics
Hemophilia B (Christmas disease)
X-Linked recessive
Locus Xq27
34 Kb of DNA 8 exons
less severe than Hemophilia A
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Molecular Pathology:
Hemophilia B
Complete/partial gene deletion
Point Mutation (67 %):
These patients
splicing
have defective mRNA
Short insertions (7 %)
Deletion (3 %)
Rarely Patients: Anti IX factor
antibody
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Antibodies that inactivate
Factor IX in response to
treatment
• Hemophilia B:
– Incidence: 50 %
• Deletions/rearangements
– Incidence: 20 %
• frameshift
• Premature stop
• Splice-site mutations
– Incidence: 0.0 %
• Missense mutation
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Inhibitors
Less common in hemophilia B
than hemophilia A
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Mutations in Hemophilia
Nonsense Mutation
Truncated Protein
Severe Hemophilia
Chromosome Inversion (about 45%(
Intron 22 (Xbal)
Severe Hemophilia
Pointmutation in CG sequence
Hot spot
Missense mutation
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Carrier detection
Intragenic RFLPs
Closely extragenic RFLPs
For E. BclI, Xbal (Fac.VIII)
For E. XmnI, TaqI (Fac.IX)
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GENETIC LINKAGE
• Mendel's original experiments
• "law of independent assortment"
• genes are transmitted from parents
to offspring independently of one
another
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Haplotype
• Any combination of genotypes can
occur
• The particular combination present in
a given individual is called a haplotype
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Inheritance of linked genes
d
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RESTRICTION FRAGMENT
LENGTH POLYMORPHISMS
• Genes can be mapped by linkage studies
with polymorphic markers
• which are nucleotide sequences identifiable
at specific sites along the genome
• Numerous markers have been identified
throughout the genome using restriction
endonucleases
• and so it is possible to construct maps of
disease genes in relation to closely linked
markers
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THE POLYMERASE CHAIN REACTION - PCR
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The restriction enzyme Hind III
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Distance between these two genes is
about 8 centi-Morgans
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MOLECULAR HYBRIDIZATION IS
USED TO DETECT SPECIFIC GENES
• 1. Single-stranded DNA is generated
• 2. A probe is a known sequence of part of
gene to be identified tagged with a
radioactive label
• Specific probes are synthesised in the
laboratory
• 3. The probe hybridizes only to the
fragment with the corresponding sequence
• This is detected by the label , which gives
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fluorescent signal
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MOLECULAR HYBRIDIZATION IS
USED TO DETECT SPECIFIC
GENES
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DNA Extraction from blood
Hemophilia A
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PCR Analysis from BclI
Hemophilia A
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PCR - Running
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Xbal Polymorphism (Factor VIII-Intron 22)
in a family with Hemophilia A
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Sequencing
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Prenatal Diagnosis
(PND)
PND (10.-16. w.):
CVS (Chorionic Villus Sampling)
AF (Amniotic Fluid)
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CVS (10.-12. weeks)
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AF (15. -16. weeks)
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First Treatment
Factor VIII / IX
Purify/ Prophylactic factor VIII / IX
from donor plasma:
Again bleeding
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Again enlarged knee joint
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The major drawback of
donor derived factor VIII
Contamination by virus
Hepatitis B/C
Human Immune deficiency Virus
(HIV)
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Second Treatment
Recombinant factor
Advantage:
No possibility of viral contamination
Disadvantage:
Generate inhibitor antibody (in
minority of patients)
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Gene Therapy
Hemophilia B
Factor IX-containing Adenoassociated virus vector
Injection in to skeletal muscle
Very small increase in factor IX level
(about 1%)
The requirement f. factor IX Infusion:
fell by 50-80% f. 3 Months
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Gene Therapy
Hemophilia B
No untoward effects such as:
Germline transmission
Anti body again factor IX
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Exceptions in hemophilia A
Germ line Mosaicism
Manifesting heterozygotes
(5% of heterozygous females: Low
level of F. VIII)
Mild Hemophilia
Mobile Elements (Transposons)
LINEs: From Ch X to Ch. 22
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Von Willebrand
disease (VWD)
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VWD
• In 1926 a Finnish physician, Dr von
Willebrand, discovered a clotting
disorder
• It is not sex linked
• is found in one form or another in
over 30 breeds of dogs
• It is not hemophilia but it's the most
commonly inherited bleeding disease
of both people and animals
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Von Willebrand
Autosomal dominant disorders
Highly variable in expression
Incidence: 1:10 000
Chromosome 12q
As a carrier protein for factor VIII
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Disorders of platelet function
• Functional disorders of platelets are
relatively rare
• most of these disorders are mild and
may not be recognized early in life
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The hallmark of von
Willebrand disease
• defective adhesive glycoprotein von
Willebrand factor
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protein VWf
•
•
•
•
•
This factor is a large
multimeric protein
Synthesized
Processed
and stored in the the endothelial
cells
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von Willebrand protein
• acts as a carrier
• stabilizer of coagulation factor VIII:
– From premature photolytic degradation
– Concentrates it at site of injury
• In the absence of vWf, the factor VIII
level is low
• In classic hemophilia A, the factor VIII
level is low because of a deficiency of
factor VIII itself
• whereas in von Willebrand disease, the
factor VIII level is low because of a
deficiency in its carrier protein
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von Willebrand disease
• relatively mild bleeding disorder
• except in the occasional patient who is
homozygous for the defect
• The bleeding manifestations are
predominantly skin-related (ie, easy
bruising, epistaxis)
• Most bleeding episodes occur following
trauma or surgery
• In women, menorrhagia is common
• Pregnant patients with this disease usually
do not have problems
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Variants of von Willebrand disease
• the common form of von
Willebrand disease (type I)
– a quantitative deficiency of
vWfA
• A common variant (type IIA)
results from:
– functionally defective vWf that
is unable to form multimers
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• Type III von Willebrand disease is a
severe form of von Willebrand
disease:
– characterized by very low levels of
vWf
– and clinical features similar to
hemophilia A
– but with autosomal recessive
inheritance
– It results from a homozygous state
or double heterozygosity
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-/y
+/+
+/y
-/+
+/y
+/y
-/y
+/y
-/+
• An RFLP was found to be associated with the X-linked
recessive disorder hemophilia A in a particular family.
Individual II-1 is affected. Assuming that no recombination
has occurred, what is the status of the individuals in
generation III with respect to hemophilia A?
a. III-1 and III-3 are affected, III-2 is normal, and III-4
is a carrier.
• b. III-1 and III-3 are affected, III-2 is normal, and III-4
is not a carrier.
• c. III-1 and III-3 are normal, III-2 is affected, and III-4
is a carrier.
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• d.
III-1, III-2, and III-3 are affected, and III-4 is a61
carrier.
Sickle Cell disorder
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Sickle Cell disorder
 Stuck the red cell in the vessels
 In children: Spleen, chest, wrists,ankles
 In adults: hips and shoulders
 Anemia (Hb 7-8 g/dl)
 Infections (take antibiotics)
 Painful crises (6-18 months)
 Swollen and inflamed (hand/food
syndrome)
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What are the
Complications?
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•
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•
•
•
•
pain episodes
increased infections
bone damage
yellow eyes or jaundice
early gallstones
lung blockage
kidney damage and loss of body water in urine
painful erections in men (priapism)
blood blockage in the spleen or liver
(sequestration)
• eye damage
• low red blood cell counts (anemia)
• delayed growth
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Prenatal diagnosis
 I. ARMS-PCR (one mutation)
 II. PCR-RFLP (one inf. RFLP)
 III. RDB (one mutation)
 IV. Sequencing
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ARMS-PCR
1
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3
4
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6
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12 13
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Doesn't cause any
health problem






α+ / º Thalassemia/Thalassemia
α+ / º Thalassemia / HbC
α+ / º Thalassemia / HbD
α+ / º Thalassemia / HbE
α+ / º Thalassemia / HbO Arab
α+ / º Thalassemia / HbS
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Other combinations





HbC / Thalassemia (no problem)
HbD / Thalassemia (no problem)
HbE / Thalassemia (serious anemia)
Hbs / Thalassemia (intermediate-severe)
HPFH* / Thalassemia (no problem)
*Heriditary persistance of fetal hemoglobin
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serious anemia
 HbH / α+/º Thalassemia
 HbS /  Thalassemia
 HbS / HbC
 HbS / HbD
 HbS / HbE
 HbS / O Arab
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Toxic protein alterations
• Usually missense mutations:
– Cause structural alteration in mono- or
oligomeric proteins
– Disrupt normal function
– Lead to toxic products or precursors
• Sickle cell mutations (hem S, b6Glu>Val)*
• * Although : recessive
• Coinheritance in cis (hem S+ b23Val>Ile)
– Sickling to manifest in the heterozygote!
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Prenatal Diagnosis (PND)
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Grateful





Mrs.Rahmani, Manijeh (MS)
Mrs.Attaran, Elham (MS)
Mrs.Taeb, Fatemeh (MS)
Mr. Babr-Zadeh, Farbod (MS)
Mr. Sajedi-Far, MohammadMehdi (BS)
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