CHIMERISM' Principles and practise'

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Products of haemopoiesis
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ABNORMALITIES IN THE HEMOPOIETIC SYSTEM

• CAN LEAD TO
• HEMOGLOBINOPATHIES
• HEMOPHILIA
• DEFECTS IN HEMOSTASIS/THROMBOSIS
• HEMATOLOGICAL MALIGNANCY

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MUTATIONS AND DNA

• VARIOUS TYPES OF MUTATIONS CAN OCCUR LEADING TO
  DISEASE PHENOTYPE
• POINT MUTATIONS
• INSERTIONS OR DELETIONS
• TRANSLOCATIONS
• COMPLEX CHROMOSOMAL REARRANGEMENTS

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Sickle cell disease
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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.

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• The ß-like globin chains are controlled by a gene
  cluster on chromosome 11 in which the different
  genes are arranged in the order
  5-e-G?-A?-?ß-d-ß-3.
• The a-like gene cluster is on chromosome 16,
  p13.3, and the genes are arranged in the order
  5-?-??-?a2- ?a1-a2-a1-?-3.

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Temporal globin expression
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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.

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Genetics of Thalassemia
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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 ß0 in which no ß globin chains are
  produced and ß, in which some ß 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.

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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 ß-thalassemia major.
• Combination of hematological and molecular
  techniques offers the most reliable and accurate
  strategy for ß-thalassemia 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.

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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
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Hemophilia Mutations

• Deletions
• Point mutations
• Flip tip mutations

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F8B
A
CEN
TEL
B
TEL
E1 E22 E23
E26
CEN
F8A
C
E1
E22
E23 E26
TEL
CEN
INVERSION 22
THE IVS 22 MUTATION IN HAEMOPHILIA A.
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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

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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.

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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

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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.

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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

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Relative Risk of Venous Thrombosis

• Normal Risk 1
• Use of OCP 4
• FVL heterozygous 5-7
• OCP 30-35
• Homozygous 80
• OCP gt100
• Prothrombin heterozygous 3
• OCP 16

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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

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Chronic Myeloid Leukaemia

• Malignancy of the haemopoietic system
• Transformation of the pluripotent stem cell
• 922 translocation giving rise to the
  Philadelphia (Ph) chromosome
• Creation of a leukaemia specific mRNA (BCR-ABL)
• Resistance to apoptosis, abnormal signalling and
  adhesion

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Clinical Course Phases of CML
Advanced phases
Chronic phase Median 46 yearsstabilization
Accelerated phase Median durationup to 1 year
Blastic phase (blast crisis) Median survival36
monthsTerminal phase
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Cytogenetic Abnormality of CMLThe Ph Chromosome
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2
3
4
5
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7
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10
11
9
12
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14
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16
17
18
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20
21
22
x
Y
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The Ph Chromosome t(922) Translocation
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9 q

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Ph ( or 22q-)
bcr
bcr-abl
abl
FUSION PROTEINWITH ELEVATED TYROSINEKINASE
ACTIVITY
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bcr-abl Gene and Fusion Protein Tyrosine Kinases
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9
Philadelphia chromosome
t(922) Translocation
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bcr
bcr-abl fusion gene
abl
ALL
p190 bcr-abl
CML
p210 bcr-abl
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Prevalence of the Ph Chromosome in
Haematological Malignancies

• Leukaemia of Ph Patients
• CML 95
• ALL (Adult) 1530
• ALL (Paediatric) 5
• AML 2

Faderl S et al. Oncology (Huntingt).
199913169-184.
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P210 stimulates signal transduction in CML cells
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Imatinib
Farnesyl transferase inhibitors (SCH 66336)
Wortmannin LY294002
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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

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MLL Promiscuous partner
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AML1

• 21q
• AML1-ETO t(821)
• T(321)
• TEL-AML t(1221)
• Loss of transactivation domain critical to
  t(821) and t(321) abnormalities
• Inv (16)

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Molecular Mechanisms of AML1 action
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AML1

• 21q
• AML1-ETO t(821)
• T(321)
• TEL-AML t(1221)
• Loss of transactivation domain critical to
  t(821) and t(321) abnormalities
• Inv (16)

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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

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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

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What do translocations involving AML1 do?

• T(821) Generates AML1-ETO fusion
• T(321) generates AML1-EVI1, AML1-EAP1 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

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Molecular Mechanisms of AML1 action
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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

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Inversion 16 and AML
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Molecular Mechanisms of AML1 action
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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

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APML MOLECULAR GENETICS

• M3 FORM OF AML
• NON RANDOM CHROMOSOMAL ABNORMALITY
• t(1517) IN 95 OF CASES
• RARa GENE ON CHROMSOME 17
• PML GENE ON CHROMOSOME 15
• t(1117) t(517)

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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

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ALL TRANS RETINOIC ACID
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NON HODGKINS LYMPHOMA

• B CELL FOLLICULAR LYMPHOMA
• t(1418)(q21q14)
• BCL 2 AND IMMUNOGLOBULIN GENES INVOLVED
• DYSREGULATION OF BCL 2
• FAILURE OF APOPTOSIS

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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