Molecular Biology and Next Generation Sequencing Meet Hematology. Ninette Amariglio Hematology Laboratory Sheba Cancer Research Center

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1 Molecular Biology and Next Generation Sequencing Meet Hematology Ninette Amariglio Hematology Laboratory Sheba Cancer Research Center July 30, 2015

2 Molecular Biology ביולוגיה מולקולרית 2

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4 The Central Dogma of Molecular Biology Cell Transcription Translation Ribosome DNA mrna Polypeptide (protein) Timothy G. Standish

5 Watson & Crick the secret of life Watson: a zoologist, Crick: a physicist In 1947 Crick knew no biology and practically no organic chemistry or crystallography.. Applying Chagraff s rules and the X-ray image from Rosalind Franklin, they constructed a tinkertoy model showing the double helix Their 1953 Nature paper: It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material. Watson & Crick with DNA model Rosalind Franklin with X-ray image of DNA 5

6 DNA, RNA, and the Flow of Information Replication Transcription Translation 6

7 DNA Chains are Long Polynucleotides The entire human genome (one complete set of the 23 chromosomes) contains about 3 billion base pairs. Each cell contains about 2 meters of DNA (1 m per chromosome set). 7 There are about cells in the human body. 2 x m is the length of about 100 trips to the sun and back!

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9 Gene Content and Genome Size of Various Organisms

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11 Large part of the mammalian genome contains repetitive sequences 11

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15 Types of Repetitive Sequences in the Human Genome Tandem repeats: satellite DNA Interspersed repeats LINEs SINEs Duplicated genes incl. pseudogenes 15

16 Simple sequence repeats Tandem repeats of a particular k-mer bp repeat unit (conserved) repeats (variable) variable numbers of tandem repeats VNTRs 3% of genome Distribution centromeres, telomeres, certain chromosomal positions. Used in mapping 16

17 17 Simple sequence repeats resulting in polymorphisms

18 Long Interspersed Nuclear Elements (LINEs) Characteristics 60 bp - 7 kb Considerable variation in length due to 5 truncations, deletions, and rearrangements ~500,000 elements/haploid genome (15-20%) 3,000-4,000 are full-length 1-2% capable of transposition, probably via an RNA intermediate (as with retroviruses) Distribution Found in A-T rich regions 18

19 Short Interspersed Repetitive Elements (SINEs): Alu Elements Characteristics Consensus: 281 nt 1 Consists GGCCGGGCGC of two GGTGGCTCAC related units GCCTGTAATC CCAGCACTTT 41 Considerable GGGAGGCCGA GGCGGGCGGA variation in length TCACGAGGTC due to AGGAGATCGA deletions, substitutions, or insertions 81 GACCATCCCG GCTAAAACGG TGAAACCCCG TCTCTACTAA ~1,000,000 elements/haploid genome (~12%) 121 AAATACAAAA AATTAGCCGG GCGTAGTGGC GGGCGCCTGT 161 AGTCCCAGCT ACTTGGGAGG CTGAGGCAGG AGAATGGCGT 201 GAACCCGGGA GGCGGAGCTT GCAGTGAGCC GAGATCCCGC 241 CACTGCACTC CAGCCTGGGC GACAGAGCGA GACTCCGTCT 281 C Distribution Average spacing is 4-kb apart Scattered but non-random? Deleterious when inserted within a gene Examples of insertions which assist in transcription regulation when inserted in control region of a gene. 19

20 Pseudogenes and Gene Fragments Many eukaryotic genes exist as variants which, for example, may be expressed during different stages of development Families of evolutionarily-diverged genes with related functions Pseudogenes: Nonfunctional gene copies or gene fragments which have arisen during gene family expansion. Contain insertions, deletions, nonsense mutations. 20 Usually non-transcribed. May be associated with functional gene copy.

21 -globin Region on Human Chromosome 11 2 G A 1 Alu repeats 10 kb 21 LINEs

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27 Epigenetic writers, readers and erasers mutated or translocated in hematologic malignancies. Chun Yew Fong et al. Haematologica 2014;99: by Ferrata Storti Foundation

28 All Cells have common Cycles Born, eat, replicate, and die 29

29 Schematic of the cell cycle. outer ring: I = Interphase,M = Mitosis ; inner ring: M = Mitosis,G = 1Gap 1,G = 2Gap 2,S = Synthesis not in ring: G = 0Gap 0/Resting. 30

30 Overview of DNA to RNA to Protein A gene is expressed in two steps 1) Transcription: RNA synthesis 2) Translation: Protein synthesis 31

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32 RNA RNA is similar to DNA chemically. It is usually only a single strand. T(hymine) is replaced by U(racil) Some forms of RNA can form secondary structures by pairing up with itself. This can change its properties dramatically. DNA and RNA can pair with each other. 33

33 RNA, continued Several types exist, classified by function mrna this is what is usually being referred to when a Bioinformatician says RNA. This is used to carry a gene s message out of the nucleus. trna transfers genetic information from mrna to an amino acid sequence rrna ribosomal RNA. Part of the ribosome which is involved in translation. 34

34 Non-coding RNAs or RNAs come more than in three flavours...

35 Of all RNA, transcribed in higher eukaryotes, 98% are never translated into proteins Of those 98%, about 50-70% are introns The rest originate from non-protein genes, including rrna, trna and a vast number of other non-coding RNAs (ncrnas) Even introns have been shown to contain ncrnas, for example snornas It is thought that there might be 10,000 different ncrnas in mammalian genome

36 The two main classes of ncrnas Housekeeping ncrnas, which are constitutively expressed and required for normal function and viability of cell Regulatory ncrnas are expressed only in certain stages of organism development or as a response to external stimuli. Regulatory ncrnas can affect the expression of other genes at the level of transcription or translation

37 Housekeeping ncrnas trna and rrna - translation snrna Pre-mRNA splicing snorna rrna modification grna guide RNA in RNA editing Telomerase RNA primer for telomeric DNA synhesis A few other...

38 39 Non-coding RNAs: the small nuclear and small nucleolar RNAs typically function as ribonucleoprotein (RNP) complexes

39 40 Regulatory RNAs - ncrnas

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45 Mouse models with impact on B-cell lymphoma development Copyright 2015 Wolters Kluwer Health, Inc. All rights reserved. Published by Lippincott Williams & Wilkins, Inc. 3

46 FIGURE 1. Copyright 2015 Wolters Kluwer Health, Inc. All rights reserved. Published by Lippincott Williams & Wilkins, Inc. 4

47 Philadelphia Chromosome 48 NEJM. 1999; 341:164

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51 Amplification of the Exogenous Internal Positive Control (IPC) Template (50-5x10 8 copies) 52 REAL TIME (RT) QUANTITATIVE PCR

52 Polymerization 5 3 Forward Primer R TaqMan Probe Q Reverse Primer 53

53 Displacement 5 3 Forward Primer R Q Reverse Primer 54

54 Cleavage R Q

55 Polymerization Completed R Q

56 Druker, B. J. Blood 2008;112: Copyright 2008 American Society of Hematology. Copyright restrictions may apply.

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59 Sensitivity of BCR/ABL kinase domain mutations to tyrosine kinase inhibitors Baccarani, M. et al. Haematologica 2008;93: Copyright 2008 Ferrata Storti Foundation

60 A. T315I mutation B. WT 61

61 SEQUENOM MassARRAY System

62 ABL kinase mutation results by Sequenom WT T315I mutation WT

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70 Blanche P. Alter Fanconi anemia and the development of leukemia Best Practice & Research Clinical Haematology, Volume 27, Issues 3 4, 2014,

71 Blanche P. Alter Fanconi anemia and the development of leukemia Best Practice & Research Clinical Haematology, Volume 27, Issues 3 4, 2014,

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78 Properties of transcription factors Generally have nuclear localization domain. Generally have a modular structure of interconnected functional domains. Two broad classes of transcription factors 79 Bind directly to the DNA. Co-factors.

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80 Mouse Rhesus Human Embryonic stem cells (ESCs)

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84 Skewed repertoire Monoclonal repertoire Polyclonal repertoire

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88 89 Mutations vs. Polymorphisms

89 The Good, the Bad, and the Silent Mutations can serve the organism in three ways: The Good : The Bad : A mutation can cause a trait that enhances the organism s function: Mutation in the sickle cell gene provides resistance to malaria. A mutation can cause a trait that is harmful, sometimes fatal to the organism: Huntington s disease, a symptom of a gene mutation, is a degenerative disease of the nervous system. The Silent: A mutation can simply cause no difference in the function of the organism. Campbell, Biology, 5 th edition, p

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102 A search for mutations by sequencing of multiple genes

103 104 The Encyclopedia of DNA Elements (ENCODE) was started in 2003, even before the first complete human genome had been published.