Week 1 CytoHistory-ChromosStruct 2024 PDF

Molecular Cytogenetics & Diagnostics Welcome to Cytogenetics! Ross Graham [email protected] Mahony Fenn [email protected] Keea Inder-Smith Molecular Cytogenetics & Diagnostics Welcome to Cytogenetics! Ross Graham [email protected] Mahony Fenn [email protected] Recomme...

Molecular Cytogenetics & Diagnostics Welcome to Cytogenetics! Ross Graham [email protected] Mahony Fenn [email protected] Keea Inder-Smith Molecular Cytogenetics & Diagnostics Welcome to Cytogenetics! Ross Graham [email protected] Mahony Fenn [email protected] Recommended texts You do not have to purchase the following textbooks but you may like to refer to them. Highly recommended Gardner RJM, Sutherland GR, Shaffer LG (2012): Chromosome Abnormalities and Genetic Counselling. 4th edition, Oxford University Press. The following textbooks are optional and contain useful information for some parts of the unit. There is no need to purchase any of them, but you may wish to refer to them. Gersen SL & Keagle MB, Eds (2013): Principles of Clinical Cytogenetics. 3rd edition, Springer. Miller OJ & Therman E (2001): Human Chromosomes. 4th edition, Springer, New York Strachan T & Read AP (2010): Human Molecular Genetics. 4th edition, Garland Science Barch M, Knutsen T & Spurbeck JL, Eds (1997): The AGT Cytogenetics Laboratory Manual. 3rd Edition Lippincott-Raven Press. Philadelphia Shaffer LG, McGowan-Jordan J & Schmid M, Eds (2013): ISCN 2013: An International System for Human Cytogenetic Nomenclature. Karger, Basel. Journals and Journal articles American Journal of Human Genetics Clinical Genetics Human Genetics Prenatal Diagnosis Citations for articles and useful references will be given to you throughout the semester. © RM Graham 2024 Assessment Summary Assessment Worth Due Mid-semester test (weeks 1-7) 20% 15th Apr Practical Assessment (3 components) Continuous 2x karyotyping tests Microarray Report 20% 10% 10% ongoing 22nd April / 6th May 13th May Final Exam (weeks 1 - 13) 40% exam fortnight TOTAL 100% © RM Graham 2024 Teaching Week 1: 26th Feb Lecture Topic History of Cytogenetics / The Human Genome 1 2 Lecturer Laboratory Topic (marks) RMG Karyotyping 1 – Introduction No classes - Labour Day holiday 3: 11th Mar Chromosome Structure / Aneuploidy MF Karyotyping 2 – Normal (2) 4: 18th Mar Structural Abnormalities / Segregation MF Karyotyping 3 – Abnormal (2) 5: 25th Mar Tissue Culture Techniques / Cytogenetics Methods MF Blood Harvest (3) 6: 1st Apr 7: 8th Apr Non-teaching week / Easter MF Banding of Chromosomes (3) MF Karyotyping 4 Microarray report information RMG Karyotyping test 1 (5) Using Genome Databases 1 (6) Case Study Information Malignancies RdK Fluorescence in situ hybridisation [TBA] Using Genome Databases 2 (Mid-semester test feedback) Genetic Couselling Examples in Cytogenetics AR MF Karyotyping test 2 (5) Completion of Database pracs 12 :13th May History of Genomics Epigenetics Linkage Analysis DMG Completion of Microarray Report (10) Case study preparation 13: 20th May The Human Genome 2 RMG Case Study (4) 8: 15th April 9: 22nd April 10: 29th April 11: 6th May 27 May - 14 Jun Sex chromosome abnormalities / Uniparental disomy Mid-semester Test [weeks 1-7] Microarrays Databases Databases tutorial Study Break/Exams © RM Graham 2024 24:1-19 (2018) © RM Graham 2024 Molecular Cytogenetics & Diagnostics History of Cytogenetics Ross Graham [email protected] Why study the history of Cytogenetics? "Those who don't know history are destined to repeat it." Edmund Burke Encyclopaedia Britannica © RM Graham 2024 Introduction to Cytogenetics Cytogenetics is a routine clinical tool Important research tool Prenatal investigations Constitutional studies Infertility Recurrent foetal loss Spontaneous miscarriage Leukaemia Solid tumour evaluation © RM Graham 2024 Introduction to Cytogenetics Cytogenetics is about cellular genetics “the scientific study of the genetics of cells; spec. the study of the structure and function of chromosomes” Oxford English Dictionary - Cyto: “of a cell or cells” - Genetics: “the study of heredity and the variation of inherited characteristics” - Cytogenetics: using chromosomes to investigate heredity Chromosomes have been identified as the vehicle of heredity © RM Graham 2024 Chromosome Structure Revision © RM Graham 2024 Female Carrion Crow Roslik GV & Kryukov AP. Rus J Genet, 2001;37:796–806. © RM Graham 2024 Female Mouse http://www.pathology.washington.edu/galleries/cytogallery/main.php?file=mouse%20karyotype © RM Graham 2024 Male Human courtesy: Mahony Fenn © RM Graham 2024 History 1950s was the birth decade of human cytogenetics Gestation: - 1909 onwards: Thomas Hunt Morgan & students - crossing-over - st 1 spontaneous mutation reported - Association of physical distance with genetic distance - Mutagenic activity of ionising radiation http://ihm.nlm.nih.gov/luna/servlet/view/all © RM Graham 2024 Cell Culture 1885: First cells kept alive in culture 1907: In vitro proliferation of cells (Harrison) 1913: Continuous culture of chick embryo cells (Carrel) - lasted until 1946... allegedly 1952: First immortalised cell line (Gey) © RM Graham 2024 Cell Culture 1952: First immortalised cell line (Gey) http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Image-Gallery/Image-Detail.9502.html © RM Graham 2024 Human Chromosome Studies 1888: Chromosomes named (Waldeyer) 1912: - Human ! 48 chromosomes - Human " 47 chromosomes (von Winiwarter) 1921: Y chromosome discovered (Painter) 1952: Improved views of chromosomes by adding hypotonic solution to cell preps (Hsu) 1953: Barr bodies identified as being X-chromosomes (Barr) © RM Graham 2024 Human Chromosome Studies 1956: Excellent views of human embryonic mitoses (Tjio) - clear that humans have 46 chromosomes Tjio & Levan: The chromosome number of man. Hereditas, 42:1-6, 1956. 1956: Confirmed 46 chromosomes in human somatic cells (Ford & Hamerton) © RM Graham 2024 Definitions Aneuploidy: an abnormal number of chromsomes Trisomy: 3 copies of a chromosome Monosomy: 1 copy of a chromosome Triploidy: 3 copies of all chromosomes © RM Graham 2024 Chromosome Abnormalities 1959: Down Syndrome caused by trisomy 21 (Lejeune, Gautier & Turpin) 1959: Turner Syndrome caused by missing X chromosome (Ford) 1959: Kleinfelter Syndrome caused by XXY 1959: Most miscarriages caused by abnormal chromosome numbers (Hertig) © RM Graham 2024 Chromosome Abnormalities 1960: Chronic Myeloid Leukaemia caused by the Philadelphia chromosomes 1963: First inherited deletion syndrome identified - “Cri du Chat”. Part of 5p is missing (Lejeune) 1963: Retinoblastoma caused by a deletion in 13q 1968: Duffy blood group located to a gene on chromosome 1 because of its association with an anomaly near the centromere of chromosome 1 www.nature.com/scitable/content/using-fish-to-detect-chromosomal-abnormalities-in-19533 © RM Graham 2024 Cytogenetic Techniques 1968: Chromosome bands created (Caspersson) Late 1960s: Collection of foetal cells via amniocentesis becomes possible 1971: Currently used convention for naming chromosome bands was introduced 1979: Flow Cytometry adapted for use in Cytogenetics © RM Graham 2024 Metaphase Chromosomes G-bands Human Molecular Genetics (4th ed); Figure 2.14 © RM Graham 2024 Cytogenetic Techniques 1982: Introduction of FISH to cytogenetics (Ward & van der Ploeg) 1985: FISH first used in humans to locate a gene 1992: CGH introduced (Kallionieni, Pinkel & Gray) 1996: Spectral karyotyping developed allowing all chromosomes to be viewed using a different colour 1998: Array-CGH increases sensitivity and throughput © RM Graham 2024 Interphase FISH Charcot-Marie Tooth Syndrome Chronic Myeloid Leukaemia: BCR-ABL http://www.nature.com/scitable/content/using-fish-to-detect-chromosomal-abnormalities-in-19533 © RM Graham 2024 Spectral Karyotyping www.cyto.purdue.edu/cdroms/micro1/7_spon/chroma/image4.htm “Talking” Genetics Glossary. J Am Med Assoc. 1999;281:600. © RM Graham 2024 Spectral Karyotyping Landry JJM et al G3: Genes|Genomes|Genetics 2013. © RM Graham 2024 Molecular Cytogenetics & Diagnostics Genome Organisation Ross Graham [email protected] The Human Nuclear Genome Nuclear DNA Single-copy functional genes Functional Sequences Families of coding genes (&related pseudogenes) Non-coding functional sequences Dispersed gene families Tandem gene families Spacer DNA Repetitive DNA Sequences with no known function Repeats in centromeric heterochromatin VNTRs Transposons Transposon sequences Retrotransposons © RM Graham 2024 Chromosome Structure Revision fibres fibre fibre. fibre fibre... © RM Graham 2024 Chromosome Structure Revision © RM Graham 2024 Chromosome Structure Revision © RM Graham 2024 A 3D Map of the Human Genome © RM Graham 2024 Chromosome Structure Revision © RM Graham 2024 Chromatin complex and intricately organised composed of DNA, proteins, RNA & polysaccharides two types: - euchromatin - heterochromatin © RM Graham 2024 Euchromatin mostly unique sequence DNA mostly coding genes loosely coiled contains active, early replicating genes stains lightly in G-banding © RM Graham 2024 Heterochromatin mostly repetitive DNA contains few active genes replicates late in S-phase highly condensed located mainly at centromeres two types: - constitutive - facultative © RM Graham 2024 Constitutive Heterochromatin located at centromeres of all chromosomes and at distal end of Y chromosome contains no transcribed genes variation in this has no effect on phenotype CH in chromosomes 1, 9, 16 and Y is of variable size functions in the regulation of crossing over © RM Graham 2024 Facultative Heterochromatin caused by inactivation of one X chromosome in every female cell Barr bodies www.mun.ca/biology/scarr/Barr_Bodies.html inactivated X is highly condensed replicates late in S-phase © RM Graham 2024 Telomeres at the end of each chromosome protective cap prevents chromosomes from joining together acts to limit cell replication (Hayflick limit) can be rebuilt by telomerase © RM Graham 2024 Telomeres © RM Graham 2024 Centromeres part of the kinetochore a constriction visible in metaphase chromosomes point at which sister chromatids are joined together consists of constitutive heterochromatin point of attachment for spindle microtubules during mitosis and meiosis © RM Graham 2024 Nucleolar Organising Regions aka “NOR” located on the satellite stalks on the short arms of acrocentric chromosomes (13, 14, 15, 21, 22) contain multiple clustered copies of genes for 18S & 23S sites of synthesis of ribosomal material during interphase not all are active can be stained using silver nitrate © RM Graham 2024 Cell Cycle the sequence of events from one cell division to the next needed for growth, repair and maintenance of cells very different in prokaryotes and eukaryotes - reflects the difference in complexity and structure between the cell types also different in animals and plants - reflects different cell wall © RM Graham 2024 Cell Cycle Two phases Interphase M phase Gap 1 (G1) Mitosis (M) Synthesis (S) Cytokinesis Gap 2 (G2) © RM Graham 2024 Cell Cycle © RM Graham 2024 Cell Cycle Two phases Interphase M phase Gap 1 (G1) Mitosis (M) Synthesis (S) Cytokinesis Gap 2 (G2) © RM Graham 2024 Cell Cycle Gap 1 (G1) starts straight after previous cell division is complete cytoplasmic components are constructed cell doubles in size lasts several hours (typically 10h for mammals) G0 this is a holding stage not all cells enter G0 cell is viable, but does not divide on exit from G0, cell can enter G1 or apoptosis pathway © RM Graham 2024 Cell Cycle G1 / S checkpoint holds the cell in G1 until everything is ready to proceed to S once past this point, the cell is committed to divide Synthesis (S) a duplicate copy is made of each chromosome each chromosome becomes two sister chromatids typically lasts 9h in mammals © RM Graham 2024 Cell Cycle Gap 2 (G2) cell prepares for mitosis mitochondria divide spindle fibre precursors are synthesised typically lasts 4h in mammals G2 / M checkpoint holds the cell if its DNA is damaged © RM Graham 2024 Cell Cycle Two phases Interphase M phase Gap 1 (G1) Mitosis (M) Synthesis (S) Cytokinesis Gap 2 (G2) © RM Graham 2024 Cell Cycle Mitosis (M) aka “karyokinesis” two daughter cells are created daughter cells are identical to the parent cell five stages - these are gradual, not distinct - prophase - prometaphase - metaphase - anaphase - telophase © RM Graham 2024 Cell Cycle © RM Graham 2024 Cell Cycle Number of molecules © RM Graham 2024 Cell Cycle Cytokinesis cell division may be simultaneous with telophase details of the process are different between cell types © RM Graham 2024 Cell Cycle Cell cycle video Mitosis video © RM Graham 2024 Cell Cycle Meiosis needed for sexual reproduction allows shuffling of genetic information important to Cytogeneticists as a source of abnormalities → 4 daughter cells, each with half the genetic information two stages - meiosis I - meiosis II © RM Graham 2024 Cell Cycle Meiosis homologues © RM Graham 2024 Cell Cycle Prophase I chromosomes leptotene pachytene zygotene diplotene pachytene diakinesis (beginning of metaphase I) Human Molecular Genetics, 2010 (4th ed); Figure 2.6 © RM Graham 2024 Cell Cycle Meiosis © RM Graham 2024