Molecular HematologyMolecular Hematology

Normal somatic cell has 46 chromosomes = diploid.

Ova and sperm have 23 chromosomes = haploid.

Karyotype shows the chromosomes from a mitotic cell in numerical order.

Aneuploid: A somatic cell with more or less than 46 chromosomes is termed

Hyperdiploid: More than 46 chromosomes

Hypodiploid : Less than 46 chromosomes.

Pseudodiploid: 46 chromosomes but with rearrangements.

Each chromosome has two arms: short arm = p and long arm = q.

Centromere: Short and long arms meet at the.

Telomeres: Ends of the chromosomes .

Each arm is divided into regions numbered outwards from the centromere.

Each region is divided into bands.

or shows loss or gain of the chromosome.

del : part of the chromosome is lost, e.g. del(16q).

Add: additional material has replaced part of a chromosome.

t: Translocation e.g. t(9; 22)

inv (inversion); part of the chromosome runs in the opposite direction.

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

Review of Major ConceptsReview of Major ConceptsCell CycleCell CyclePoint MutationPoint Mutation

– Occurs when a single nucleic acid base is changed, resulting in either missence or nonsense Occurs when a single nucleic acid base is changed, resulting in either missence or nonsense mutationmutation

Protein MalformationProtein MalformationDNA Transcription FactorsDNA Transcription Factors

– Endogenous substances, usually proteins, which are effective in the initiation, stimulation, or Endogenous substances, usually proteins, which are effective in the initiation, stimulation, or termination of the genetic transcription process termination of the genetic transcription process

Tumor-Suppressor GenesTumor-Suppressor Genes Genes that inhibit expression of the tumorigenic phenotype. When tumor suppressor Genes that inhibit expression of the tumorigenic phenotype. When tumor suppressor

genes are inactivated or lost, a barrier to normal proliferation is removed and genes are inactivated or lost, a barrier to normal proliferation is removed and unregulated growth is possible.unregulated growth is possible.

OncogenesOncogenes -Genes which can potentially induce neoplastic transformation. They include genes for growth -Genes which can potentially induce neoplastic transformation. They include genes for growth factors, growth factor receptors, protein kinases, signal transducers, nuclear phosphoproteins, and factors, growth factor receptors, protein kinases, signal transducers, nuclear phosphoproteins, and transcription factorstranscription factors. .

Good prognosisGood prognosis–Hyperdiploidy >50Hyperdiploidy >50–ETV6-CBFA2 (tel-aml)ETV6-CBFA2 (tel-aml)

t(12;21)t(12;21)

–E2A-PBX1E2A-PBX1t(1;19)t(1;19)

Poor prognosisPoor prognosis–BCR-ABLBCR-ABL

t(9;22)t(9;22)

–MLL rearrangementsMLL rearrangementst(4;11), t(11;19),t(1;11)t(4;11), t(11;19),t(1;11)

Genetics of Haematological MalignanciesHaematological malignancies are mostly clonal disorders resulting from a genetic alteration.

Genes involved : oncogenes and tumour-suppressor genes.

Normal proliferation and apoptosis Excess proliferation / loss of apoptosis

Tumour-suppressorgene

OncogeneProto-oncogene

Tumour-suppressorgene

Oncogenes

Oncogenes result from gain-of-function mutations of proto-oncogenes that would normally control the activation of genes.

Translocation may lead to:

(a) over-expression of an oncogene under the control of the promoter of another gene, e.g. an immunoglobulin or T cell receptor gene as seen in lymphoid malignancies.

(b) fusion of segments of two genes creating a novel fusion gene and thus a fusion protein, e.g. in CML.

Tumour-Suppressor Genes

Tumour-suppressor genes are subject to loss-of-function mutations (point mutation or deletion) and thus malignant transformation.

Tumour-suppressor genes help regulate cells to pass through different phases of the cell cycle, e.g. G1 to S, S to G2 and mitosis.

Clonal Progression

Malignant cells may acquire new characteristics resulting from new chromosomal changes causing acceleration.

Multidrug resistance (MDR) is one complication. The cells may start to express a protein which actively pumps the chemotherapeutic agent to the outside of the cells.

p53 Proteinp53 ProteinOne gene one monomer One gene one monomer Its consists of 4 different Its consists of 4 different monomersmonomersIf one of the monomers is If one of the monomers is dysfunctional the whole protein dysfunctional the whole protein becomes defunctbecomes defunctThus all it takes its one mutant Thus all it takes its one mutant gene for the protein to become gene for the protein to become defunctdefunctCytosol levels rise rapidly in Cytosol levels rise rapidly in response to DNA damaging agentsresponse to DNA damaging agentsIf damage is found in the template If damage is found in the template or complementary strand then or complementary strand then duplication stopsduplication stopsThe amount of p53 will stop The amount of p53 will stop Synthesis in the cell cycleSynthesis in the cell cycleIf it reaches a threshold level then If it reaches a threshold level then it induces the cell to undergo it induces the cell to undergo apoptosisapoptosisEvolutionary homology with Evolutionary homology with murines, reptiles, even yeastmurines, reptiles, even yeast

P53 monomer

Causes of leukemiaCauses of leukemia??????Clonal expansionClonal expansion a cell that has the a cell that has the ability to self-replicate but unable to ability to self-replicate but unable to differentiatedifferentiateGeneticsGenetics– Higher incidence in siblings and twinsHigher incidence in siblings and twins

VirusVirus– Clusters of leukemiaClusters of leukemia

Ionizing radiationIonizing radiation– Survivors of Hiroshima and NagasakiSurvivors of Hiroshima and Nagasaki

Syndromes with higher Syndromes with higher incidenceincidence

Down’sDown’s

Bloom’sBloom’s

Fanconi’sFanconi’s

Klinefelter’sKlinefelter’s

Ataxia Ataxia telangiectasiatelangiectasia

Methods Used to Study the Genetics of Malignant Cells

1-Karyotype Analysis(Cytogenetic studies)Images of chromosomes are captured when cell is in metaphase.

2-Immunofluorescence StainingCan be useful for a few chromosomal abnormalities, e.g. promyelocytic leukaemia protein which normally has a punctate distribution but is diffusely scattered in acute promyelocytic leukaemia with the t(15; 17) translocation. Abnormal fusion proteins may also be detected by specific monoclonal antibodies.

3-Fluorescent in situ Hybridisation (FISH)Fluorescent-labelled genetic probes hybridise to specific parts of the genome. Can pick up extra copies of genetic material in both metaphase and interphase, e.g.trisomy 12 in CLL. Translocations can be seen by using two different probes.

4-Southern Blot AnalysisRestriction enzyme digestion of DNA, gel electrophoresis and “blotting” to a suitable membrane. DNA fragments are hybridised to a probe complementary to the gene of interest. If the probe recognises a segment within the boundaries of a single fragment one band is identified. If the gene has been translocated to a new area in the genome a novel band of different electrophoretic mobility is seen.

5-Polymerase Chain Reaction (PCR)Can identify specific translocations, e.g. t(9; 22). Can also detect clonal cells of B- or T-cell lineage by immunoglobulin or T-cell receptor (TCR) gene rearrangement analysis. Sensitivity (can detect one abnormal cell in 105–106 normal cells) makes this of value in monitoring patients with minimal residual disease (MRD).

6-DNA Microarray PlatformsRapid and comprehensive analysis of cellular transcription by hybridising labelled cellular mRNA to DNA probes immobilised on a solid support. Oligonucleotides or complementary DNA (cDNA) arrays are immobilised on the array and fluorescent labelled RNA from the cell sample is annealed to the DNA matrix. Can determine the mRNA expression pattern of different leukaemia subtypes.

ThalassemiasThalassemias

Thalassemias are a heterogenous Thalassemias are a heterogenous group of genetic disordersgroup of genetic disorders– Heterozygous individuals exhibit varying Heterozygous individuals exhibit varying

levels of severitylevels of severity– The disorders are due to mutations that The disorders are due to mutations that

decrease the rate of synthesis of one of decrease the rate of synthesis of one of the two globin chains (the two globin chains ( or or ). The ). The genetic defect may be the result of:genetic defect may be the result of:

ThalassemiasThalassemiasA mutation in the noncoding introns of the gene resulting A mutation in the noncoding introns of the gene resulting in inefficient RNA splicing to produce mRNA, and therefore, in inefficient RNA splicing to produce mRNA, and therefore, decreased mRNA productiondecreased mRNA production

The partial or total deletion of a globin geneThe partial or total deletion of a globin gene

A mutation in the promoter leading to decreased A mutation in the promoter leading to decreased expressionexpression

A mutation at the termination site leading to production of A mutation at the termination site leading to production of longer, unstable mRNAlonger, unstable mRNA

A nonsense mutationA nonsense mutation– Any of these defects lead to:Any of these defects lead to:

An excess of the other normal globin chainAn excess of the other normal globin chain

A decrease in the normal amount of physiologic A decrease in the normal amount of physiologic hemoglobin madehemoglobin made

Development of a Development of a hypochromic, microcytic anemiahypochromic, microcytic anemia

ThalassemiasThalassemiasThe clinical expression of the different gene The clinical expression of the different gene combinations (1 from mom and 1 from dad) are as combinations (1 from mom and 1 from dad) are as follows:follows:

00//00, , +1+1/ / +1+1, or , or 00/ / +1,+2,or +3 +1,+2,or +3 = thalassemia major, the = thalassemia major, the most severe form of the disease.most severe form of the disease.

Imbalanced synthesis leads to decreased total RBC Imbalanced synthesis leads to decreased total RBC hemoglobin production and a hypochromic, hemoglobin production and a hypochromic, microcytic anemia.microcytic anemia.Excess Excess chains precipitate causing hemolysis of chains precipitate causing hemolysis of RBC precursors in the bone marrow leading to RBC precursors in the bone marrow leading to ineffective erythropoiesisineffective erythropoiesisIn circulating RBCs, In circulating RBCs, chains may also precipitate chains may also precipitate leading to pitting in the spleen and decreased RBC leading to pitting in the spleen and decreased RBC survival via a chronic hemolytic process.survival via a chronic hemolytic process.The major cause of the severe anemia is the The major cause of the severe anemia is the ineffective erythropoiesisineffective erythropoiesis..

ThalassemiasThalassemias– Beta (Beta () thalassemia) thalassemia

The disease manifests itself when the switch from The disease manifests itself when the switch from to to chain synthesis occurs several months after birth chain synthesis occurs several months after birthThere may be a compensatory increase in There may be a compensatory increase in and and chain synthesis resulting in increased levels of hgb F chain synthesis resulting in increased levels of hgb F and Aand A22. The genetic background of . The genetic background of thalassemia is thalassemia is heterogenous and may be roughly divided into two heterogenous and may be roughly divided into two types:types:

00 in which there is complete absence of in which there is complete absence of chain chain production. This is common in the Mediterranean.production. This is common in the Mediterranean.

++ in which there is a partial block in in which there is a partial block in chain synthesis. chain synthesis. At least three different mutant genes are involved:At least three different mutant genes are involved:

+1+1 – 10% of normal – 10% of normal chain synthesis occurs chain synthesis occurs+2+2 – 50% of normal – 50% of normal chain synthesis occurs chain synthesis occurs+3+3 - > 50% of normal - > 50% of normal chain synthesis occurs chain synthesis occurs

The clinical applications of thrombophilia The clinical applications of thrombophilia susceptibility genes.susceptibility genes.

Susceptibility GeneSusceptibility Gene Clinical ApplicationClinical Application

•Prothrombin Mutation:G20210A. •Factor V Leiden Mutation: R506Q.

  Hereditary thrombophilia

•Platelet GP Ia Mutation: •C807T and 648A (HPA-5). * Platelet GP IIIa: Mutation:T393C(HPA-la/b=P1Al/P1A2)

   Bleeding tendency due to Bleeding tendency due to platelets dysfunctionplatelets dysfunction

*Factor IX propeptide Mutations at ALA-10

Coumarin hypersensitivityCoumarin hypersensitivity