Download CHIMERISM. Principles and practise.

Products of haemopoiesis
ABNORMALITIES IN THE
HEMOPOIETIC SYSTEM
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CAN LEAD TO
HEMOGLOBINOPATHIES
HEMOPHILIA
DEFECTS IN
HEMOSTASIS/THROMBOSIS
• HEMATOLOGICAL MALIGNANCY
MUTATIONS AND DNA
• VARIOUS TYPES OF MUTATIONS CAN
OCCUR LEADING TO DISEASE
PHENOTYPE
• POINT MUTATIONS
• INSERTIONS OR DELETIONS
• TRANSLOCATIONS
• COMPLEX CHROMOSOMAL
REARRANGEMENTS
Sickle cell disease
Thalassemia
• The thalassemias are a diverse group of genetic blood
diseases characterized by absent or decreased production
of normal hemoglobin, resulting in a microcytic anemia of
varying degree
• The alpha (a) thalassemias are concentrated in
Southeast Asia, Malaysia, and southern China.
• The beta (b) thalassemias are seen primarily in the areas
surrounding Mediterranean Sea, Africa and Southeast Asia.
The β-like globin chains are controlled by a gene cluster on chromosome 11 in which
the different genes are arranged in the order 5’-ε-Gγ-Aγ-ψβ-δ-β-3’.
The α-like gene cluster is on chromosome 16, p13.3, and the genes are arranged in the
order 5’-ζ-ψζ-ψα2- ψα1-α2-α1-θ-3’.
Temporal globin expression
Temporal Globin expression
a globin expression is rather stable in fetal and adult life,
because it is needed for both fetal and adult hemoglobin
production
b globin appears early in fetal life at low levels and rapidly
increases after 30 weeks gestational age, reaching a
maximum about 30 weeks postnatally
g globin molecule is expressed at a high level in fetal life ( 6
weeks) and begins to decline about 30 weeks gestational
age, reaching a low level about 48 weeks postgestational
age.
d globin appears at a low level at about 30 weeks gestational
age and maintains a low profile throughout life.
Genetics of Thalassemia
Types of Thalassemia
 b thal: excess of a globins, leading to formation of a globin
tetramers (a4) that accumulate in the erythroblast , leading to
ineffective erythropoiesis. Two types of mutations, the β0 in
which no β globin chains are produced and β+, in which some
β chains are produced but at a reduced rate.
 a thal : excess of b globins, leading to the formation of b
globin tetramers (b4) called hemoglobin H. Results in
hemolysis, generally shortening the life span of the red cell.
Hemoglobin H-Constant Spring disease is a more severe form
of this hemolytic disorder. Most severe form is a thalassemia
major, in which fetus produces no a globins, which is generally
incompatible with life.
Thalassemia Prevention
• Preventive programs in (i) public education, (ii) population
screening, genetic counseling and prenatal diagnosis have
been very effective in reducing the birth rate of βthalassemia major.
• Combination of hematological and molecular techniques
offers the most reliable and accurate strategy for βthalassemia prenatal diagnosis
• Development of molecular techniques not only made it
possible to offer prenatal diagnosis at an early stage of the
pregnancy but they can help to resolve diagnostic
problems.
HAEMOPHILIA
X LINKED RECESSIVE DISORDER
HAEMOPHILIA A – MUTATIONS IN FACTOR VIII GENE
HAEMOPHILIA B – MUTATIONS IN FACTOR IX GENE
SIMPLE AND COMPLICATED MUTATIONS
THE FLIP TIP MUTATION
Hemophilia Mutations
• Deletions
• Point mutations
• Flip tip mutations
F8B
A
E1
E22
E23 E26
CEN
TEL
F8A
B
TEL
E1
E22
E23 E26
CEN
F8A
C
E22
E1
E23 E26
TEL
CEN
INVERSION 22
THE IVS 22 MUTATION IN HAEMOPHILIA A.
Activated Protein C and Factor V
• The function of protein C is to inactivate factor Va and
factor VIIIa
• The first step in this process is the activation of
thrombomodulin by thrombin. Subsequently, protein C
combines with thrombomodulin in order to produce
activated Protein C (APC)
• Activated protein C can then degrade factor Va and factor
VIIIa
Factor V Leiden
• Factor V Leiden is a genetically acquired
trait that can result in a thrombophilic
(hypercoaguable) state resulting in the
phenomenon of activated protein C
resistance (APCR)
• Over 95% of patients with APCR have
factor V Leiden.
Activated Protein C and Factor V
Leiden
• When one has factor V Leiden, the factor Va is resistant to
the normal effects of activated protein C, thus the term
activated protein C resistance
• The result is that factor V Leiden is inactivated by
activated protein C at a much slower rate (see Figure 3),
thus leading to a thrombophilic (propensity to clot) state by
having increased activity of factor V in the blood
Prevalence of FVL
• Factor V Leiden is seen more commonly in the northern
European populations
• About 4-7% of the general population is heterozygous for
factor V Leiden. About 0.06 to 0.25% of the population is
homozygous for factor V Leiden.
• The factor V Leiden mutation is relatively uncommon in
the native populations of Asia, Africa and North America.
In contrast, in Greece and southern Sweden, rates above
10% have been reported.
Prothrombin and Deep Vein
Thrombosis
• Prothrombin is the precursor to thrombin in the
coagulation cascade
• Thrombin is required in order to convert fibrinogen into
fibrin, which is the primary goal of the coagulation cascade
• The gene has a mutation at position 20210, hence the
disorder being referred to as prothrombin mutation 20210
• The prothrombin gene mutation is seen more commonly in
the Caucasian population. About 1-2% of the general
population is heterozygous for the prothrombin gene
mutation
Relative Risk of Venous
Thrombosis
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Normal Risk
Use of OCP
FVL heterozygous
+ OCP
Homozygous
+ OCP
Prothrombin heterozygous
+ OCP
1
4
5-7
30-35
80
>100
3
16
Leukaemia, the current
hypothesis
• Defect in maturation of white blood cells
• May involve a block in differentiation and/or a
block in apoptosis
• Acquired genetic defect
• Initiating events unclear
• Transformation events involve acquired genetic
changes
• Chromosomal translocation implicated in many
forms of leukaemia
Chronic Myeloid Leukaemia
• Malignancy of the haemopoietic system
• Transformation of the pluripotent stem cell
• 9;22 translocation giving rise to the
Philadelphia (Ph’) chromosome
• Creation of a leukaemia specific mRNA
(BCR-ABL)
• Resistance to apoptosis, abnormal signalling
and adhesion
Clinical Course: Phases of CML
Advanced phases
Chronic phase
Median 4–6 years
stabilization
Accelerated phase
Blastic phase (blast crisis)
Median duration
up to 1 year
Median survival
3–6 months
Terminal phase
Cytogenetic Abnormality of CML:
The Ph Chromosome
1
6
2
7
3
8
13
14
19
20
4
9
15
21
5
10
16
22
11
17
x
12
18
Y
The Ph Chromosome:
t(9;22) Translocation
9
9 q+
22
Ph ( or 22q-)
bcr
bcr-abl
abl
FUSION PROTEIN
WITH ELEVATED
TYROSINE
KINASE ACTIVITY
bcr-abl Gene and Fusion Protein Tyrosine Kinases
9+
9
Philadelphia
chromosome
22
bcr
t(9;22) Translocation
bcr-abl
fusion gene
abl
Chromosome 9
Chromosome 22
bcr
1
13
14
ALL
CML
breakpoint breakpoint
c-abl
1
2-11
p190 bcr-abl
ALL
p210 bcr-abl
CML
Prevalence of the Ph Chromosome
in Haematological Malignancies
Leukaemia
% of Ph+ Patients
CML
95
ALL (Adult)
15–30
ALL (Paediatric)
5
AML
2
Faderl S et al. Oncology (Huntingt). 1999;13:169-184.
P210 stimulates signal transduction
in CML cells
Imatinib
Farnesyl
transferase
inhibitors
(SCH 66336)
Wortmannin
LY294002
ACUTE LEUKEMIA
• Translocation is a major mechanism
• Involves genes whose normal function is to
control cell division, haematological
development etc
• These genes are known as master genes
• MLL, AML1
• Mutatation of these genes through
translocation leads to leukemia
MLL Promiscuous partner
AML1
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21q
AML1-ETO t(8;21)
T(3;21)
TEL-AML t(12;21)
Loss of transactivation domain critical to
t(8;21) and t(3;21) abnormalities
• Inv (16)
Molecular Mechanisms of AML1
action
AML1
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21q
AML1-ETO t(8;21)
T(3;21)
TEL-AML t(12;21)
Loss of transactivation domain critical to
t(8;21) and t(3;21) abnormalities
• Inv (16)
What is AML1
• Subunit of a multifactorial transcription factor
known as Core Binding Factor
• AML1 is also known as Core Binding FactorA
• It has homology to the drosophila developmental
gene “runt” in its DNA binding region
• Also has a transactivation domain at its carboxy
terminus
What does AML 1 do?
• Binds DNA
• Binding site for AML1 is a core enhancer that is
located at the 5’ control region of genes that are
involved in controlling lineage differentiation
• T cell receptor , myeloperoxidase, IL3, GM-CSF,
CSF1
• AML1 plays a pivotal role in hemopoietic
differentiation by orchestrating expression of
appropriate lineage specific genes
What do translocations involving
AML1 do?
• T(8;21) Generates AML1-ETO fusion
• T(3;21) generates AML1-EVI1, AML1EAP1 or AML1-MDS1
• All of the above involve replacement of the
transactivation domain
• These new fusion proteins can no longer
activate AML1 binding sites in lineage
specific genes
Molecular Mechanisms of AML1
action
Inversion 16
• Here AML1 is not involved
• However the other member of the Core
Binding Factor complex (CBFb) is mutated
• Net result is a pertubation of transcription of
genes with AML1 binding sites
Inversion 16 and AML
Molecular Mechanisms of AML1
action
Summary
• Master genes such as AML1 and MLL
control lineage specific gene expression,
thus orchetrating lineage specific
development of hemopoiesis
• Mutations in these genes disrupt this
control, thus leading to aberrant
hemopoiesis and development of leukemia
APML MOLECULAR
GENETICS
• M3 FORM OF AML
• NON RANDOM CHROMOSOMAL
ABNORMALITY
• t(15;17) IN 95% OF CASES
• RARa GENE ON CHROMSOME 17
• PML GENE ON CHROMOSOME 15
• t(11;17); t(5;17)
MOLECULAR MEDICINE
INTO ACTION
• PRESENCE OF RARa CRITICAL TO THE
TREATMENT OF THIS DISEASE
• STANDARD CHEMOTHERAPY ONLY
PARTIALLY EFFECTIVE
• TREATMENT WITH RA REMOVES
DIFFERENTIATION BLOCKADE
ALL TRANS RETINOIC ACID
NON HODGKINS
LYMPHOMA
• B CELL FOLLICULAR LYMPHOMA
• t(14;18)(q21;q14)
• BCL 2 AND IMMUNOGLOBULIN
GENES INVOLVED
• DYSREGULATION OF BCL 2
• FAILURE OF APOPTOSIS
Summary
• Molecular changes implicated in
haemoglobinopathies
• Factor VIII and Factor IX in Haemophilia
• Factor V leiden and Prothrombin in Deep vein
Thrombus
• Molecular abnormalities in Leukemia, particularly
translacations
• CML, a paradigm for malignancy
• Mutations in master genes disrupt control of
hemopoiesis leading to development of leukemia
• Knowledge of molecular changes can influence
diagnosis, prognosis and treatment