AS.Molecular Biology in Hematologic disease.ppt

Agus SuronoOctober 2010

Cytogenetic Genetics Diagnostic Engineering Gene therapy

2Kuliah Biomol 2010

3Kuliah Biomol 2010

Human Genome ProjectsHuman Genome Projects

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Regulation of gene : Regulation of gene :

DNA

mRNA

Nucleus

Cytoplasm

Protein

Intracellular

Extracellular

Membran cell

Transcription

Translation

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DNADNA RNARNA ProteinProtein

transcription translation

Protein SynthesisProtein Synthesis

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Nucleus Cytoplasm

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Protein maturationProtein maturation

Postranslational modification- glycosilation- phosphorylation

Folding

Resite

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Origin

Copy ????

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Autosomal Dominant Autosomal Recessive X-Linked Multifactorial

Identification/DiagnosticIdentification/Diagnostic


The haploid human genome has 3 X 109 base pairs of DNA sequence

This contains approximately50,000 - 100,000 genes

(more?)



14FISH: Fluorescence In Situ Hybridization

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Single Copy DNA (60%) Mainly Euchromatic regions Genes, Pseudogenes

Repetitive DNA (40%) Mainly Heterochromatic regions Tandem repeats

Microsatellite repeats Minisatellite repeats

Interspersed repeats


Identify large families with the disease

Clinical Assessment.

Identify a chromosomal locusLinkage.

Refine the candidate intervalRun more markers, construct a contig.

Identify Candidate GenesDatabases, exon trapping, direct selection.

Mutation AnalysisSequencing, southern blotting, SSCP, ....


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INTRODUCTION OF INTRODUCTION OF HEMATOLOGYHEMATOLOGY

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Cellular Components Components of Blood

Red Blood Cells White Blood Cells Inflammatory process Coagulation

Hemorrhage Blood Typing Clotting disorders Transfusion Medicine


SpleenSpleen

KidneysKidneys

LiverLiverBoneMarrowBone

Marrow

BloodBlood

HematopoieticSystem

HematopoieticSystem


Pluripotent Stem Cell

Myeloid Multipotent Stem Cells

Common LymphoidStem Cells

Unipotent Progenitors

BasophilsEosinophilsNeutrophilsMonocytes

Lymphocytes

ErythrocytesThrombocytes

Erythropoietin

WBC’sRBC’s

Platelets20Kuliah Biomol 2010

Plasma Transport

mechanism 90-92% water. 6-7% proteins 2-3%

Fats Carbohydrates

(glucose) Electrolytes Gases (O2, CO2) Chemical

messengers

Plasma Components

Other3%

Protein7%

Water90%


Red Blood Cells Erythrocyte

Hemoglobin – O2 bearing molecule Comprised of 4

subunits: Globin (binds to 1

O2 molecule) Heme (iron)

100% saturation = 4 globin subunits carrying O2

Each gram of hemoglobin = 1.34 ml O2

Cellsalive.com

Components of BloodComponents of Blood


Red blood cell production: Erythropoiesis

Erythropoietin

Hemolysis Sequestration Laboratory analysis of

red blood cells Red blood cell count Hematocrit Hemoglobin



Platelets (Thrombocytes) Megakaryocytes

Thrombopoietin Thrombocytopenia Thrombocytosis



Class IClass I Class IIClass II Class IIIClass III Class IVClass IV

Blood Loss (ml)Blood Loss (ml) Up to 750Up to 750 750-1500750-1500 1500-20001500-2000 >2000>2000

Blood Loss (% Blood Loss (% volume)volume)

Up to Up to 15%15%

15%-30%15%-30% 30%-40%30%-40% >40%>40%

PulsePulse <100<100 >100>100 >120>120 >140>140

BPBP NormalNormal NormalNormal DecreasedDecreased DecreasedDecreased

Pulse PressurePulse Pressure Normal or Normal or increasedincreased

DecreaseDecreasedd

DecreasedDecreased DecreasedDecreased

Resp. RateResp. Rate 14-2014-20 20-3020-30 30-4030-40 >35>35

Urine Output Urine Output (ml/hr)(ml/hr)

>30>30 20-3020-30 5-155-15 NegligibleNegligible

Mental StatusMental Status Slightly Slightly anxiousanxious

Mildly Mildly anxiousanxious

Anxious, Anxious, confusedconfused

Confused, Confused, lethargiclethargic

Fluid Fluid Replacement Replacement (3:1) rule(3:1) rule

CrystalloiCrystalloidd

CrystalloiCrystalloidd

Crystalloid Crystalloid and bloodand blood

Crystalloid Crystalloid and bloodand blood


Blood Types Antigens

A, B, AB, O Rh factor

Rh+ = ~85% Rh- = ~15%

Blood transfusion


Blood Typing - ABOBlood Typing - ABO

Blood typeBlood type Antigen Antigen present on present on erythrocyteerythrocyte

Antibody Antibody present in present in

serumserum

OO

ABAB

BB

AA

NoneNone

A and BA and B

BB

AA

Anti-A, Anti-BAnti-A, Anti-B

NoneNone

Anti-AAnti-A

Anti-BAnti-B27Kuliah Biomol 2010

Compatibility Among ABO Blood GroupsCompatibility Among ABO Blood Groups

Reaction with serum of RecipientReaction with serum of RecipientDonor CellsDonor Cells ABAB BB AA OO

ABAB

BB

AA

OO

--

--

--

--

++

--

++

--

++

++

--

--

++

++

++

---= no reaction+ = reaction

Blood Products and Blood Blood Products and Blood Typing Typing


Brady; Paramedic Care Principles and Practice

Blood Products and Blood Blood Products and Blood Typing Typing


Membrane defect of erythrocytes:


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The Red Cell

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Eritrosit

Hb

Heme + Globin

Fe + Porfirin Chain + non

(,,)

bilirubin

300 juta/sel

SINTESIS Hb & HEMOLISISSINTESIS Hb & HEMOLISIS

Protein (integral, peripheral)

Strength, flexibility (deformability) erythrocytes

Bilayer lipid membrane:

- Protein, lipid, karbohidrat (50,40,10%) - Phospholipid - Attach to cytoskeleton


Protein transmembrane (integral)

- Band 3 (ABO, I, i)

- Glycophorine A (M, N, P. falciparum), B (S, s, U), C- Protein Rh

Function: transport, pump, signal transduction

Protein cytoskeleton (peripheral)

Spectrin , β (band 1,2), actin, ankyrin, band 4.1, band 4.2

Cell stability function: vertikal : spectrin, band 3, ankyrin, protein 4.2 horizontal: spectrin, actin, protein 4.1

Hereditary Spherocytosis

Pathophysiology:

Defect spectrin

Cell membrane is not well constructed

Permeability NaK, dehidration

LIEN‘conditioning effect’

Sferis, fragil

DestructionM, fisik

HEMOLISIS


Band Protei BM /sel Kromosom Gena AA Gena Penyakit

1 Spectri 240240 242242 1q22q231q22q23 80 (Kb)80 (Kb) 24292429 SPTA1SPTA1 HE,HPP,HS,NIHF

2 β Spectri 220220 242242 14q22p24.214q22p24.2 >100>100 21372137 SPTBSPTB HE,HPP,HS,NIHF

2.1 Ankyrin 210210 124124 8p11.28p11.2 >120>120 18801880 ANK1ANK1 HS

2.9 Adduci 103 103 3030 4p16.34p16.3 -- 737737 ADDAADDA -

3* AnExch1 9090 12001200 17q12p2.117q12p2.1 1717 911911 EPB3EPB3 HS,SAO,NIHF

4.1 P4.1 8080 200200 1p33p34.21p33p34.2 >250>250 588588 EL1EL1 HE

4.2 P4.2 7272 250250 15q15q2115q15q21 2020 691691 ELB42ELB42 HS

4.9-8

PAS1 GlypA 3636 -1 rb-1 rb 4q314q31 >40>40 131131 GYPAGYPA

PAS2 GlypC 3232 -200-200 2q144212q14421 1414 128128 GYPCGYPC HE

PAS3 GlypB 2020 -200-200 4q314q31 >30>30 7272 GYPBGYPB

*protein integral; HPP: Hereditary pyropoikilocytosis; NIHF: Nonimmune hydrops fetalis; SAO: Southeast Asian Ovalocytosis

Gene defect protein defect Clinically: hemolytic


Anemia Anemia is a sign, not a separate disease

process. Signs and symptoms may not be present

until the body is stressed. Differentiate chronic anemia from acute

episode. Treat signs and symptoms.

Maximize oxygenation and limit blood loss.

Establish IV therapy if indicated.


Brady; Paramedic Care Principles and Practice

Diseases of Diseases of ErythrocytesErythrocytes


Sickle Cell Disease Normal red cells maintain

their shape as they pass through the capillaries and release oxygen to the peripheral tissues (upper panel). Hemoglobin polymers form in the sickle cell cells with oxygen release, causing them to deform. The deformed cells block the flow of cells and interrupt the delivery of oxygen to the tissues (lower panel).



Sickle Cell Disease (cont.) Sickle cell crises

Vaso-occlusive Musculoskeletal/abdominal pain Priapism Renal/cerebral infarctions

Hematological Lowered hemoglobin Splenic sequestration

Infectious

Management Follow general treatment guidelines prn. Consider analgesics.



Polycythemia Overproduction of erythrocytes.

Occurs in patients > 50 years old or with secondary dehydration.

Most deaths due to thrombosis Results in bleeding abnormalities:

Epistaxis, spontaneous bruising, GI bleeding.

Management: Follow general treatment guidelines.



THALASSEMIA

HEMOGLOBINOPATHY

Sandoz Atlas: Cinical Hematology, 1988 Gower Medical Publishing 43Kuliah Biomol 2010

Eritrosit

Hb

Heme + globin

Fe + Porfirin Rantai + non (,,)

ThalassemiaThalassemia

300 juta/sel

Sintesis hemoglobinSintesis hemoglobin

2 2 = Hb A >96%2 2 = Hb A2

<3,5%2 2 = Hb F <2%

Molekul Hb (tetramer)

H H eemmee


Pathophysiology: (Thalassemia )

Defect sintesis of globin

Deformability/liability

Fragilities

LIEN

DestructionM, physic

HEMOLISIS

‘excessive’ of globins Deposited in cell

membrane


Sp band3 crosslinking

Abnormal Spetcrin association

Abnormal Sp low binding

Deficiency of Spect, Ank Abnormal Sp 4.1 binding

Sp Actin,4.1 instability

Lipid bilayer damage

Excess globin chain Degradat

ion

Denaturation

Band S clustering

Mechanichal removal

Hypercoagulability

Immune removal

“Excessive” of globin result in:

Thalassemia


Leukopenia/Neutropenia Too few white blood cells or

neutrophils. Follow general treatment guidelines

and provide supportive care. Leukocytosis

An increase in the number of circulating white blood cells, often due to infection. Leukemoid reaction


Leukemia Cancer of hematopoietic cells Initial presentation

Acutely ill, fatigued, febrile and weak, anemic. Thrombocytopenia Often have a secondary infection.

Management Follow general treatment guidelines. Utilize isolation techniques to limit risk of

infection.


Lymphomas Cancers of the lymphatic system

Hodgkin's Non-Hodgkins

Presentation Swelling of the lymph nodes Fever, night sweats, anorexia, weight loss,

fatigue, and pruritis

Management Follow general treatment guidelines. Utilize isolation techniques to limit risk of

infection.


Multifactorial (viral infection, etc.....)

Complex-genes involvement Chromosomal anomaly :

translocation Diversity on clinical manifestation Therapy approach gene therapy

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Thrombocytosis and Thrombocytopenia Thrombocytosis

An abnormal increase in the number of platelets

Thrombocytopenia An abnormal decrease in the number of

platelets Sequestration Destruction (ITP) Decreased production

Management Provide supportive care and follow general

treatment guidelines.

Clotting DisordersClotting Disorders


Hemophilia Deficiency or absence of a blood clotting factor

Deficiency of factor VIII causes hemophilia A. Deficiency of factor IX causes hemophilia B. Deficiency is a sex-linked, inherited disorder.

Defective gene is carried on the X chromosome.

Signs & Symptoms Numerous bruises, deep muscle bleeding, and

joint bleeding.



HEMOPHILIA

Victoria princess family, England 54Kuliah Biomol 2010

• Gene defect/protein FVIII (A), FIX (B)• Bleeding tendency (oozing)• Mild, moderated, severe•Herediter (X-linked) or spontan (approx. 30%)

Role of FVIII/IX in Role of FVIII/IX in hemostasishemostasis

Gene defect FVIII/IX

Deficiency FVIII/IX

Fibrin

Bleeding

Trombosit

Coagulationb c

Hemophilia: defect protein of coagulation factor VIII/IX

Vasokon

str

ksi

A

Hemostasis mechanism

‘close’ the hole



Gene defect FVIII http://europium.mrc.rpms.ac.uk

1. Gene rearrangement40% hemophilia A severeCross over gene fragmented (meiosis)

2. Single-base substitutions

3. Sequence deletion• >100 kb = severe,

FVIII %, undetected Ag inhibitor?

• <100 kb = mild/moderete

4. Sequence insertion

Management:Causative: factor first! (cryoprecipitate, concentrate, recombinant)

Bleeding correction (surgery)Supportive (transfusion, etc)

Preventive: Marriage counselingDiagnosis of prenatalProphylaxis of bleeding

Complication: Transfusion, inhibitor visibility, high morbidity

Hemophilia (cont.) Management

Treat the patient similarly to others. Administer supplemental oxygen. Establish IV access.

Be alert for recurrent or prolonged bleeding, and prevent additional trauma.

Von Willebrand’s Disease Deficient component of factor VIII

Generally results in excessive bleeding. Generally is not serious; provide supportive

care.




Disseminated Intravascular Coagulation System activation of coagulation cascade. Results from sepsis, hypotension, OB

complications, severe tissue or brain injury, cancer, and major hemolytic reactions.

Multiple Myeloma Cancerous disorder of plasma cells. Pathologic fractures are common.









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AS.Molecular Biology in Hematologic disease.ppt