Molecular Hematology

Galila Zaher

DNA RNA Protein

DNA & RNADNA RNADouble-stranded Single-stranded4 bases: A, C, G & T A, C, G & USugar: Deoxribose Sugar: RiboseStable molecule Unstable moleculeIntrons + Exons Introns

DNA: Building Blocks

Purine

Pyrimidine

BasesPurinesAdenine

GuaninePyrimidinesCytosine

Thymine[Uracil]

Purine ‘pair’ pyrimidine

Adenine-Thymine

Guanine-Cytosine

DNANature paper here

ChromosomesMale karyotype

46:XY

Female karyotype

46:XX

22 pairs of autosomes + 1 pair sex chromosomes

Normal StructureSomatic cell has 46 chromosomes : diploid.Ova and sperm have 23

chromosomes :haploid.Karyotype shows chromosomes of dividing

cell in numerical order.

Chromosome has two arms:

Short arm = p Long arm = q.

Short and long arms meet at the Centromere.

Ends of the chromosomes are called Telomeres

Each arm is divided into regions numbered from centromere.

Each region is divided into bands.

Numerical Abnormality Aneuploid: Somatic cell with >or <46

chromosomesA. Hyperdiploid: >46 chromosomesB. Hypodiploid : <46 chromosomes. Pseudodiploid: 46 chromosomes but with

rearrangements.

Structural Abnormalitydel : deletion where part of chromosome is lost

del(16q).Add: additional material has replaced part of a Cht: Translocation t(9; 22)inv :inversion; part of Ch runs in opposite direction.Point mutationNon sense :Result in creation of premature stop codon

Normal sequence ATG CTG TGC CysMutant sequence ATG CTG TGA stop

i: isochromosome is a chromosome with identical chromosome arms at each end, e.g. i(17q) has two copies of 17q joined at centromere.

Haematological MalignanciesMostly clonal disorders resulting from a genetic

alteration.Tumor-Suppressor Genes : inhibit expression

of tumor phenotype. When are inactivated or lost abnormal proliferation

Oncogenes :Genes which can potentially induce neoplastic transformation. They include genes for growth factors, growth factor receptors, protein kinases,etc.

Normal proliferation / ApoptosisExcess proliferation / loss of Apoptosis

Tumour-suppressorgene

OncogeneProto-oncogene

Tumour-suppressorgene

Genetics of Haematological Malignancies

Mutation

Mutation or deletion

Clonal ProgressionActivation of OncogenesInactivation of Tumour-Suppressor GenesMalignant cells acquire new characteristics

causing acceleration.Multidrug resistance (MDR) is one

complication. Cells start to express a protein which actively pumps chemotherapeutic agent to outside of cells.

Thalassemia Heterogenous group of genetic disorders

Mutation decrease rate of synthesis of globin chains ( or ).

0 :complete absence of chain . Common in Mediterranean.

+ :partial block in chain synthesis. 1. Noncoding introns inefficient RNA splicing ,decreased

mRNA production2. Promoter leading to decreased expression3. Termination site production of longer, unstable mRNA

Partial or total deletion of a globin gene thalassemia major :0/0, +/ +, or 0/ +

ß-ThalassemiasDisease manifests itself when switch chain ms

after birth Imbalanced synthesis low Hb production, MCV &

MCH Excess chains precipitate in RBC precursors in bone

marrow leading to hemolysis and ineffective erythropoiesis

Excess chains in circulating RBCs precipitate leading to pitting in spleen & RBC survival via a chronic hemolytic process.

The major cause of severe anemia is the ineffective erythropoiesis.

Compensatory increase in & chain synthesis Hb F& A2.

Hereditary thrombophiliaPCPSATProthrombin Gene Mutation:G20210A. Factor V Leiden Mutation: R506Q.MTRFR :mutation

INHERITED RISK FACTORS INHERITED RISK FACTORS Relative risk of VTERelative risk of VTE

0

20

40

50

Heterozygous deficiency of AT,

PC, PS

Heterozygous G1691A FV

Homozyougs G1691A FV

Heterozygous G20210

prothrombin

Homozyg prothrombin

10-fold 5- to 8-fold

50- to 80- fold

30

60

70

80

2-to 4- fold 10- fold

Gene Structure

Splice sites

X-linked DisordersHaemophilia A and BX-linked disordersMales affected –

females carriers

Queen Victoria (1819 - 1901)

Queen Victoria Queen of England from 1837 to 1901 was a carrier.

Her eighth child, Leopold, had Hemophilia and suffered from frequent hemorrhage.

Descendants Eugenie, who was a carrier introduced Hemophilia into Spanish royal family .

Irene married to Prince Henry of Prussia introduced the disease into the German royal family

Alexandra married Russia's last czar Nicholas II introduced the disease into the Russia royal family, which ultimately played a role in the start of the Russian Revolution.

Royal Disease

Alexis, son of Nicholas and Alexandra (1904 - 1918)

Alexandra gave birth to a son Alexis the long awaited heir Russian throne.

Unfortunately Alexis had Hemophilia which ultimately played a role in the start of the Russian Revolution.

Victoria will be remembered as the cause of Hemophilia which spread to the Royal Family of Europe through her descendants

F8 Gene

FIX Gene

Types of MutationsMissense mutationsNonsense mutationsSplice mutationsInsertionsDeletions

Inversions Sever HASHA: Intron 22: ~50% cases Intron 1: <1% cases

Haemophilia A: Intron 22 Inversion

Cytogenetic &Molecular studies• Karyotype Analysis (numerical ) • Immunofluorescence Staining

(structural)• Fluorescent in situ Hybridisation (FISH)• Southern Blot Analysis• Polymerase Chain Reaction (PCR)

t(9,22),hemophilia,thrombophilia• DNA Microarray Platforms

ChromosomesMale karyotype

46:XY

Female karyotype

46:XX

22 pairs of autosomes + 1 pair sex chromosomes