Cytogenetics 1

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Cytogenetics 1

  1. 1. Cytogenetic Parvaneh Afsharian Department of Genetics, Royan Institute
  2. 2. - - Aging -
  3. 3. 9banding - -
  4. 4. DNA  Chromosomes
  5. 5. DNA  Chromosomes
  6. 6. DNA  Chromosomes
  7. 7. Clinical Cytogenetic
  8. 8. History • Human Chromosomes; 1882 in Tumor cells by Walther Flemming (mitosis discoverer) Emery & Rimon’s principles and practice of Medical genetics (2007), Elsevier
  9. 9. History • Chromosome: introduced by von Waldeyer (1888) (colored bodies) • ≥ 50 years later , 2n= 46 Emery & Rimon’s principles and practice of Medical genetics (2007), Elsevier
  10. 10. History • 1912: Hans von Winiwater; Spermatogonia (47) & Oogonia (48) (XX/XO system) • 1922: Painter: 2n= 48 or 46 (XX/XY system) • 1956: Tjio & Levan (Hypotonic Sol)  2n= 46 in human embryonic cells Clinical Cytogenetic was born Von Winiwarter H 1912, Arch Biologie Painter TS 1922 Anat Res Painter TS 1923 J Exp Zoology Tjio JH & Levan A 1956 Hereditas
  11. 11. History • • • • • 1956: Clinical cytogenetics 1959: +21 (France), 45,XO (UK) 1960: Ph (t(9;22)) in CML 1960s end: Banding techniques  Chr. Identification 1977: ISCN Gilgenkrantz S et al 2003 The history of Cytogenetics. Annales de Genetique Garcia-Sagredo JM 2008 Biochim Biophys Acta
  12. 12. ISCN : An International System for Human Cytogenetic Nomenclature
  13. 13. Human Chromosomes: Nomenclature & Classification • Until 1970s: by size & centromer position (Group Analysis, A,B,C,D,E,F,G, Sex chromosomes)
  14. 14. Group Analysis A B C D F E G Sex chromosomes
  15. 15. Definitions • Cytogenetics – Visual study of chromosomes at microscopic level • Karyotype – Chromosome complement – also applied to picture of chromosomes • Idiogram – Stylised form of karyotype
  16. 16. Chromosomes • Classified according to position of centromere • Central centromere - metacentric • Sub-terminal centromere - acrocentric – have satellites which contain multiple copies of genes for ribosomal RNA on short arm • Intermediate centromere - submetacentric
  17. 17. chromosome identification bands numbered from 1, starting near the centromere short arm on the top, long arm on the bottom centromere location key in identification metacentric – in center; arms about equal in length submetacentric – arms unequal acrocentric – centromere near one end telocentric – centromere ‘at one end’ acrocentric – satellites on short arm
  18. 18. Banding techniques
  19. 19. Region Band Sub-Band
  20. 20. Sub-band 1p36.1 1. Metacentric 2. Sub metacentric 3. Acrocentric
  21. 21. GTG banded human chromosomes with banded cartoon along side
  22. 22. Human male G-banda
  23. 23. laboratory • Sampling (Blood: Leukocytes) • Culturing (mitogen; medium; P/S) • Harvesting [colcemid; hypotonic sol (KCl) & fixative (MeOH:CH3COOH)] • Slid spreading • Staining (aging; banding techniques)
  24. 24. Day II
  25. 25. Genetic Counseling • • • • Medical base Medical genetic knowledge Communication process Deals with the human problems associated with: - occurrence - risk of occurrence of a genetic disorder in a family - risk of recurrence •pedigree Medical Genetics for the Modern Clinician, 2006, Lippincott Williams & Wilkins
  26. 26. Pedigree
  27. 27. Inheritance patterns • Autosomal Dominant Inheritance • Autosomal Recessive Inheritance • X-Linked Dominant Inheritance • X-Linked Recessive Inheritance Medical Genetics for the Modern Clinician, 2006, Lippincott Williams & Wilkins
  28. 28. Check of your practice
  29. 29. Banding techniques
  30. 30. Chromosome staining • Q-banding – Quinacrine stain • G-banding – Giemsa stain • C-banding – heterochromatin regions which remain condensed (regions near centromere are heterochromatin) • R-banding – reverse banding • FISH – fluorescence in situ hybridization probes for specific genes or locations probes tagged with fluorescent molecules • Spectral karyotyping probes specific for each chromosome, different colors
  31. 31. special procedures C banding – staining of heterochromatin (condensed DNA) region near centromere High-resolution banding – staining of less condensed chromosome regions non-staining regions on several chromosomes – fragile sites (fragile X – mental retardation)
  32. 32. FISH
  33. 33. Spectral karyotyping
  34. 34. Spectral karyotyping
  35. 35. Array CGH • Comparative genomic hybridization (numerical Abnormalities)
  36. 36. Clinical Cytogenetics
  37. 37. Chromosomal Abnormalities
  38. 38. Chromosomal abnormalities Numerical Polyploidy (triploidy, tetraploidy) Aneuploidy (monosomy, trisomy, tetrasomy) Structual Translocations Inversions Insertions Deletions Rings Isochromosomes ESAC (Extra Structurally Abnormal Chromosome)
  39. 39. Numerical Chromosomal Abnormalities
  40. 40. Numerical Chromosomal Abnormalities-I •Euploidy or polyploidy: multiple N haploid – 1N or 23 chromosomes diploid – 2N or 46 chromosomes triploid – 3N or 69 chromosomes tetraploid – 4N or 92 chromosomes (~5% spontaneous abortions)
  41. 41. Chromosome abnormalities triploid – 3N due to ‘dispermy’ found in 15-18% of spontaneous abortions enlarged head fusion of fingers & toes malformations of mouth, eyes &genitals
  42. 42. Partial Hydatidiform mole
  43. 43. Numerical Chromosomal Abnormalities-II Add/Del. Aneuploidy: 45 or 47 chromosomes Monosomy: one of a pair missing (usually lethal) 45, X Trisomy : caused by non-disjunction XXY (47, XXY); + 21; +18
  44. 44. Aneuploidy of autosomes • Trisomy 21 – Down syndrome (47, XY, +21) 1 in 900 live births leading cause of mental retardation and heart defects phenotype distinctive skin fold near eye – epicanthic fold spots in iris – Brushfield spots wide skull, flatter than normal at the back tongue often furrowed and protruding congenital heart defects in ~40% of cases physical growth, behavior & mental development prone to respiratory infections leukemia (higher rate than normal) Maternal age is a factor Medical Genetics for the Modern Clinician, 2006, Lippincott Williams & Wilkins
  45. 45. Down syndrome, trisomy 21 47,XX,+21 or 47,XY,+21
  46. 46. Aneuploidy of autosomes • Trisomy 13 – Patau syndrome (47, XX, +13) 1 in 5,000 live births condition lethal phenotype facial malformations eye defects extra fingers or toes malformations of brain & nervous system congenital heart defects parental age is a factor Medical Genetics for the Modern Clinician, 2006, Lippincott Williams & Wilkins
  47. 47. Patau syndrome, trisomy 13 47,XX,+13 or 47,XY,+13 Incidence at birth 1/5,000
  48. 48. Patau Syndrome
  49. 49. Aneuploidy of autosomes • Trisomy 18 – Edward syndrome (47, XX, +18) 1 in 3,000 live births 80% of live births are female phenotype small at birth, grow slowly mentally retarded clenched fists; 2nd & 5th fingers overlap 3rd & 4th malformed feet; heart malformations common parental age is a factor Medical Genetics for the Modern Clinician, 2006, Lippincott Williams & Wilkins
  50. 50. Edwards syndrome, trisomy 18 47,XX,+18 or 47,XY,+18 Incidence at birth 1/3,000
  51. 51. Edward Syndrome
  52. 52. Aneuploidy of Sex chromosomes Abnormalities more tolerated • Extra X/Y Y: few genes, mostly sex determination X: excess X is inactivated • Monosomy X, Turners – – – – Majority die during development Only small proportion survive at birth Short and infertile 1 in 2500 live birth
  53. 53. Sex chromosome abnormalities • Turner Syndrome 45,XO (female) 47, XXX (female) 47,XXY (male) • Extra “Y” chromosome 47,XYY (male) 1/2500 • Trisomy X 1/1000 • Klinefelter Syndrome 1/500 1/1000 Medical Genetics for the Modern Clinician, 2006, Lippincott Williams & Wilkins
  54. 54. Structural Chromosomal Abnormalities
  55. 55. Structural Chromosomal Abnormalities – Translocations – Inversions – Insertions – Deletions – Rings – Isochromosomes – ESAC
  56. 56. Dfinitions • Rearrangements: – Deletion: a segment is lost – Duplication: a segment is doubled – Inversion: a segment within the chromosome is reversed – Translocation: a segment is moved to a different chromosome • The origin of these rearrangements can be: – Breakage and rejoining – Crossing-over between repetitive DNA
  57. 57. Crossing-over between Repetitive DNA
  58. 58. Balanced Rearrangements Change the chromosomal gene order but do not remove or duplicate any of the DNA of the chromosomes • There are two classes of balanced rearrangements: 1. Inversion: is a rearrangement in which an internal segment of a chromosome has been broken twice, flipped 180 degrees, and rejoined 2. Reciprocal translocation: is a rearrangement in which two chromosomes are each broken once, creating acentric fragments, which then trade places
  59. 59. INVERSIONS
  60. 60. 1. Paracentric: if the centromere is outside the inverted segment 2. Pericentric: if the centromere is within the inverted segment Ordinarily, no genetic material is gained or lost in an inversion Thus an individual, whether homozygous or heterozygous for the inversion, generally shows no phenotypic effect. While no genetic material is lost, if breakpoints occur within genes, can cause mutations.
  61. 61. Effects of inversions at the DNA level
  62. 62. However, there are reproductive consequences for the heterozygote. This is due to problems with pairing of homologues during meiosis.
  63. 63. The homologous chromosomes attempt to align similar regions next to each other as well as they can.
  64. 64. The chromosomes assume this characteristic loop configuration
  65. 65. This causes no problem, unless crossing over occurs within the inverted region
  66. 66. Crossing-over in paracentric inversion: (inversion does not include the centromere) Results: 1 normal chromosome 2 deletion chromosomes (inviable) 1 inversion chromosome (all genes present; viable)
  67. 67. Crossing-over in pericentric inversion: (inversion includes the centromere) Results: 1 normal chromosome 2 deletion/duplication chromosomes (inviable) 1 inversion chromosome (all genes present; viable)
  68. 68. Since the only viable offspring are those that result from gametes which did not have crossovers within the inverted region, it appears that crossing over in the inversion has been suppressed So this is referred to as crossover suppression
  69. 69. Keep in mind that crossing over actually does occur in this region We just can’t observe the result in the progeny The genetic result is very tight linkage of genes in an inverted segment
  70. 70. This may be important in the evolution of some, or many, organisms Why?
  71. 71. A species may evolve a particular set of alleles at several genes on one chromosome which make individuals possessing them very well adapted. We call this set a coadapted gene complex
  72. 72. Problem is, every generation meiosis and crossing over threaten to “break up” these coadapted gene complexes
  73. 73. However, if the coadapted gene complex is within an inversion, no recombination will occur and the alleles will travel along together generation after generation
  74. 74. TRANSLOCATIONS - Reciprocal - Robertsonian
  75. 75. Usually, the term is used for exchanges of segments between nonhomologous chromosomes These are interchromosomal translocations
  76. 76. It is also possible to have intrachromosomal translocations, in which the segment stays within the same chromosome
  77. 77. We will limit our attention to interchromosomal translocations Within these, there are 2 types: reciprocal (or balanced) and nonreciprocal
  78. 78. As with inversions, translocations usually involve no net gain or loss of genetic material
  79. 79. Because of this, there are usually no phenotypic consequences for being heterozygous. Like other chromosomal rearrangements, if breakpoints occur within genes, can result in mutation of that gene. The most frequent and important type of translocation is the reciprocal translocation.
  80. 80. Homozygotes have normal meioses. Homologues can pair properly, and crossing over poses no problems.
  81. 81. But meiosis is a problem in heterozygotes. Homologues assume a characteristic crossshape (cruciform) arrangement at metaphase Disjunction can occur in 3 ways, 2 of which produce abnormal gametes
  82. 82. Down Syndrome can arise from a Robertsonian fusion between chromosome 14 and 21. Most of chromosome 21 is translocated to chromosome 14 - can result in Familial Down syndrome.
  83. 83. Segregation of a Robertsonian Translocation C B A Gamtes always get either A or B; 50% get C.
  84. 84. Translocations can sometimes be harmful. Even though there is no gain or loss of genetic material, the change in location of a segment may alter the regulation of a gene in the segment.
  85. 85. This is especially apparent if the gene is involved in the regulation of cell division. Lack of proper regulation of such a gene can result in cancer. In which case, the gene becomes known as an oncogene
  86. 86. A good example is the translocation between chromosomes 9 and 22, creating the “Philadelphia chromosome” This causes about 90% of the cases of chronic myelogenous leukemia
  87. 87. Origin of the Philadelphia chromosome in chronic myelogenous leukemia (CML) by a reciprocal translocation involving chromosomes 9 and 22 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
  88. 88. Burkitt’s lymphoma is another example of a cancer which is usually (90%) caused by a translocation (8 and 14)
  89. 89. These are examples of the phenomenon called position effect The phenotype seen depends not just on the allele of a particular gene, but also the position of the gene in a particular chromosome
  90. 90. Unbalanced rearrangements They change the gene dosage of a part of the affected chromosome • Classes of unbalanced rearrangements: 1. All whole chromosome aneuploidies 2. Deletions 3. Duplications • The duplicated segment can end up at a different position on the same chromosome, or even on a different chromosome 4. Amplifications
  91. 91. DELETIONS
  92. 92. Deletions involve the loss of a chromosome segment Because these mutations are due to the loss of genetic material, they cannot revert to wild type
  93. 93. The effects of the deletion depend on which genes are deleted And on what alleles of these genes reside on the homologous chromosome
  94. 94. Any genes in the deleted region are now present in a hemizygous condition on the homologue
  95. 95. If these alleles are recessive, their phenotypes will now be expressed This phenomenon is called pseudodominance
  96. 96. Deletion Mapping A WT deletion B C A B D E F a X F b c d e f a E b c d e f F1: 50% 50% A B C D a b c d A B a b & E c E F e All WT f F d e f Mutant phenotype for c and d (c & d phenotype “uncovered by deletion)
  97. 97. Several human disorders are due to deletions. All of these are small deletions - large deletions apparently cannot be tolerated Also, the deletions have their effects in heterozygotes; homozygotes are probably lethal
  98. 98. Cri du chat syndrome
  99. 99. Due to a deletion of part of the short arm of chromosome 5 1/50,000 births Crying babies sound like cats; mental disability Death by about 4 years
  100. 100. DUPLICATIONS
  101. 101. A segment of chromosome is doubled A good example of duplication is seen in the Bar mutants of Drosophila
  102. 102. Different numbers of copies of the 16A region of the X chromosome
  103. 103. These duplications probably arise by the process of unequal crossing over
  104. 104. Unequal crossing-over produces Bar mutants in Drosophila
  105. 105. As the number of duplicate copies of a segment increases, the likelihood of unequal crossing over also increases Thus, once the process has started there is a tendency over evolutionary time for the number of copies to increase
  106. 106. Duplication in this way by unequal crossing over is thought to be an important process in the evolution of genes
  107. 107. If a gene is crucial to the organism, it is not free to change much It certainly cannot take on a new function, since its original one is still needed
  108. 108. But, if a new copy of the gene is produced by unequal crossing over, the extra copy can evolve over time Eventually perhaps producing a protein with very different functions
  109. 109. This kind of process can result in “families” of related genes, making similar proteins Good examples of this are the globin genes, which produce the alpha and beta globin chains which comprise hemoglobin
  110. 110. ESAC • • • • • • Extra Structurally Abnormal Chromosome Abnormal chromosome in addition to 46 Small and difficult to identify Sometimes called marker chromosomes Difficult to work out effect on person May be benign or cause serious mental handicap
  111. 111. Check of your practice
  112. 112. Chromosome Study (Analysis)
  113. 113. Karyotyping • Staining methods to identify chromosomes • • • • G banding Q banding R banding C banding - Giemsa - Quinacrine - Reverse - Centromeric (heterochromatin) • Ag-NOR stain - Nucleolar Organizing Regions (active)
  114. 114. G banding • Most common method used • Chromosomes treated with trypsin – denatures protein • Giemsa stain – – – – each chromosome characteristic light and dark bands 400 bands per haploid genome Each band corresponds to 5-10 megabases High resolution (800 bands ; prometaphase chromosome) – use methotrexate and colchicine • Dark bands are gene poor
  115. 115. G banding • • • • • • Metaphase spreads Count chromosomes in 10-15 metaphases If mosaicism suspected, count 30 Detailed analysis of 3-5 metaphases Used to photograph and cut out Now computer programmes
  116. 116. Q banding • Used especially for Y chromosome abnormalities or mosaicism • Similar pattern to G banding – But can detect polymorphisms • Needs fluorescent microscope
  117. 117. R banding • Used to identify the X chromosome abnormalities • Heat chromosomes before staining with Giemsa • Light and dark bands are reversed
  118. 118. C banding • • Used to identify centromeres / heterochromatin Heterochromatic regions – – • contain repetitive sequences highly condensed chromatin fibres Treat with chromosomes with 1. Acid 2. Alkali 3. Then G band
  119. 119. Chromosome Banding resolutions Total bands: 10+X+18q+11p Banding Resolution 15 16 17 18 19 20 21 22 350 375 400 410 420 430 440 450 32-33 36 550 610 41 700
  120. 120. Cytovision Applied imaging/Leica

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