Molecular Hematology

1. Molecular Hematology Galila Zaher

2. DNA RNA Protein

3. DNA & RNA DNARNA Double-stranded Single-stranded 4 bases: A, C, G & T A, C, G & U Sugar: Deoxribose Sugar: Ribose Stable molecule Unstable molecule Introns + Exons Introns

4. DNA: Building Blocks Bases Purines Adenine Guanine Pyrimidines Cytosine Thymine [Uracil] Purine ‘pair’ pyrimidine Adenine-Thymine Guanine-Cytosine Pyrimidine Purine

5. DNA Nature paper here

6. Chromosomes Male karyotype 46:XY Female karyotype 46:XX 22 pairs of autosomes + 1 pair sex chromosomes

7. Normal Structure • Somatic cell has 46 chromosomes : diploid. • Ova and sperm have 23 chromosomes :haploid. • Karyotype shows chromosomes of dividing cell in numerical order.

9. Chromosome has two arms: Short arm = p Long arm = q.

10. Short and long arms meet at the Centromere.

11. Ends of the chromosomes are called Telomeres

12. Each arm is divided into regions numbered from centromere.

13. Each region is divided into bands.

15. Numerical Abnormality • Aneuploid: Somatic cell with >or <46 chromosomes • Hyperdiploid: >46 chromosomes • Hypodiploid : <46 chromosomes. • Pseudodiploid: 46 chromosomes but with rearrangements.

16. Structural Abnormality • del : deletion where part of chromosome is lost del(16q). • Add: additional material has replaced part of a Ch • t: Translocation t(9; 22) • inv :inversion; part of Ch runs in opposite direction. • Point mutation • Non sense :Result in creation of premature stop codon Normal sequence ATG CTG TGC  Cys Mutant 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.

17. Haematological Malignancies • Mostly 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 potentiallyinduce neoplastic transformation. They include genes for growth factors, growth factor receptors, protein kinases,etc.

19. Genetics of Haematological Malignancies Proto-oncogene Oncogene Mutation Excess proliferation / loss of Apoptosis Normal proliferation / Apoptosis Tumour-suppressor gene Tumour-suppressor gene Mutation or deletion

20. Clonal Progression • Activation of Oncogenes • Inactivation of Tumour-Suppressor Genes • Malignant 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.

21. 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. • Noncoding introns inefficient RNA splicing ,decreased mRNA production • Promoter leading to decreased expression • Termination site production of longer, unstable mRNA • Partial or total deletion of a globin gene •  thalassemia major :0/0, +/ +, or 0/ +

22. ß-Thalassemias • Disease 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.

23. Hereditary thrombophilia • PC • PS • AT • Prothrombin Gene Mutation:G20210A. • Factor V Leiden Mutation: R506Q. • MTRFR :mutation

24. 50- to 80- fold 80 70 60 50 40 30 20 10-fold 10- fold 5- to 8-fold 2-to 4- fold 0 Heterozygous deficiency of AT, PC, PS Heterozygous G1691A FV Homozyougs G1691A FV Heterozygous G20210 prothrombin Homozyg prothrombin INHERITED RISK FACTORS Relative risk of VTE

25. Gene Structure Splice sites

26. X-linked Disorders • Haemophilia A and B • X-linked disorders • Males affected – females carriers

27. 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.

28. Royal Disease • 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.

29. 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.

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

31. F8 Gene

32. FIX Gene

33. Types of Mutations • Missense mutations • Nonsense mutations • Splice mutations • Insertions • Deletions

34. Inversions Sever HA SHA: Intron 22: ~50% cases Intron 1: <1% cases

35. Haemophilia A: Intron 22 Inversion

36. 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

37. Chromosomes Male karyotype 46:XY Female karyotype 46:XX 22 pairs of autosomes + 1 pair sex chromosomes