Cytogenetic abnormalities

cytogenetic abnormalities
dr dhanya a n
sri siddhartha medical college, tumkur

1. Cytogenetic abnormalities Presenter -Dr Dhanya A N Moderator–Dr chaithra

2. Contents Introduction to cytogenetics Milestones in cytogenetics Chromosome Cell cycle and cell division Indications for cytogenetic analysis Approach to cytogenetic analysis Chromosomal abnormalities chromosomal disorders chromosomal abnormalities in neoplasm - hematological - soft tissue tumors

3. Introduction to cytogenetic CYTOGENETICS DEFINITION : Cytogenetics is the study of chromosomes and its abnormalities: alteration in the number and structure.

4. MILESTONES IN CYTOGENETICS : Arnold: First observed chromosomes:1879 Hansemann & Flemming Counted the chromosomes: 1891 Winiwarter : Isolated X chromosome Painter: Isolated Y chromosome Tijio and Levan : 1956: Described correct chromosome number as 22 pair of autosomes and 2 sex chromosomes. Levan introduced the use of Colchicine to arrest mitosis at metaphase. Hsu,Makino & Nishimura and Hughes- Hypotonic technique: In 1952 in karyotyping . Gall and Prudue described in situ hybridization techniques.

5. Chromosome Is a packaged and organized structure containing the  DNA  of a living organism

6. Types of chromosomes

7. CELL CYCLE Interphase – G1, S, G2 M phase (Mitosis) G0 – NO cell division

8. INDICATIONS FOR CYTOGENETIC ANALYSIS Prenatal – pregnancies involving older (>35yrs) women. Confirmation or exclusion of diagnosis - known chromosomal syndromes. Unexplained psychomotor retardation with or without dysmorphic features. Abnormalities of sexual differentiation and development - ambiguous genitalia.

9. Continued.. Recurrent miscarriage, stillbirth or spontaneous abortions. Females with proportionate short stature and primary amenorrhea. Parents and children of persons with chromosomal translocations, deletions and duplications. Pregnancies at risk of aneuploidy from results of fetal ultrasound. Neoplastic conditions- soft tissues and hematological.

10. APPROACH TO THE DIAGNOSIS OF CYTOGENETIC DISORDERS Karyotyping Insitu hybridization Fluorescence insitu hybridization Spectral karyotyping Comparative genomic hybridization

11. KARYOTYPE Standard display of stained and photographed chrosmosomes in metaphase spread, arranged in pairs, in order of decreasing length . Human somatic cells - 22 pair of autosomes identical in male and female 2 sex chromosomes XX - female & XY - males.

12. TISSUE SAMPLES & CELL CULTURE: Prenatal Amniotic fluid - 20ml Chorionic villi - 25mg of Vascularised and budding villi from chorion frondosum PUBS ( percutaneous umbilical blood sampling)

13. Postnatal Peripheral blood – 4 ml of heparinised Skin fibroblasts- 4mm diameter Bone marrow- 1 ml of heparinised bone marrow. Lymph node - 0.5 to 1 cm 3 Solid tumors- Part of specimen submitted for histopathological examination. Ideally 0.5-1 g m

14. 2. Culture Culture medium – Preservative free sodium heparin , ( RPMI 1640 ) , mitotic stimulant ( phytohemagglutinin ) and antibiotics( penicillin, streptomycin ). Short term culture: 1-3 days – blood, bonemarrow , chorionic villi Long term culture: 1-3 wks - other tissue types

15. Arrest of cell division : at metaphase, by Colchicine [ Deacetyl methyl colchicine ] for 20 min. Cells harvested – centrifugation  Incubated for 10min in hypotonic solution ( dilute solution of KCl 0.075 mol) .

16. 5. Cell fixation- 3:1 methanol/ glacial acetic acid mixture ( carnoy’s ) for 30min. Staining : Trypsinization of the chromosomes prior to staining, weakens the DNA-Protein interactions, add buffer (Na2HPO4 and NaH2PO4), banding techniques done with the dyes. Microscopic analysis and photography Karyotype production (manual/automated) 9. Interpretation

17. Staining Q-banding- The first banding method developed Uses quinacrine mustard or quinacrine dihydrochloride , creates a flourescent transfers band on exposure to UV light, Q- bands fade over time not routinely used .

18. G-banding Uses Giemsa dye to produce transverse bands light (G-C rich DNA) dark band (A-T rich DNA) G-bands are identical to Q-bands G-banding is the most widely used banding technique for routine chromosome analysis Around 400 bands per haploid genome seen. Each band corresponds to 5-10megabases

19. R-banding Treating chromosomes with a hot alkaline solution before Giemsa staining Produces bands that are the reverse of G-bands, called R-bands. In R banding telomeres should appear as dark bands and their absence as the result of deletion is more obvious.

20. C-banding selectively stains constitutive heterochromatin, and are located at all centromeres and distal long arm of Y chromosome. Staining with giemsa followed by heat denaturation results in darkly staining heterochromatic regions at centromere with light staining chromosome arms.

21. Nuclear organizing region (NOR) banding Specific chromosomal region that forms and maintain the nucleoli are called NOR. NOR located on stalk of acrocentric chromosomes and contain gene for 18S and 28S rRNA . Stained by Geimsa (N- banding) or silver impregnation ( Ag-NOR)

22. Successful cytogenetic analysis depends on – - Cells must be in adequate numbers - Analysis must be performed on viable cells in division - Chromosomes must be separated from one another - Chromosomes must be identified & characterized normal/abnormal - Arranged according to the length in a decreasing order.

23. K aryotyping Chromosome from each metaphase spread are arranged in prescribed order – karyotype cells – imaged, printed & karyotyped

24. ISCN International System for Human Cytogenetic Nomenclature. Centromere divide the chromosome into short arm and long am Chromosome arms divided into regions on the basis of landmarks The region adjacent to centromere of short arm and long arm are given number 1 as p1, q1 respectively, the next distal region is given 2 and so on The regions are subdivided into bands and the bands are subdivided into sub bands as the resolution increases and the numbering done sequentially

25. ISCN of chromosome 11

26. In situ hybridization Hybridization refers to the binding or annealing of complementary DNA or RNA sequences Main purpose – detection of specific nucleic acid sequences in chromosomes. In early studies, radio isotopes were used as labels for nucleic acids, and detection of hybridized sequence were done with autoradiography.

27. As technology advanced, detection by enzymatic and fluorescent means become available for quick and safe analysis. Uses- Detection of missing,additional chromosomes,chromosome rearrangements and microdeletions .

28. Fluorescent in situ hybridization The probe and metaphase target are denatured by a high temperature and formamide . Probe is hybridized to the chromosomal target. Unbound probe is removed by post hybridization washes. Bound probe is detected by fluorescence microscopy

29. Types of probes 1] Centromere enumerating probe(CEP) - Bind to highly repitative sequence alpha satellite sequences of centromere and produce strong signals. Similar sequences in pericentric region results in cross hybridization artifact.

30. 2] Locus specific identifier(LSI) probe Target distinct chromosomal region of interest and utilize single copy rather than repetitive DNA.

31. 3] Whole chromosomes probes Also known as chromosome painting probes or chromosomes libraries , consists of thousands of overlapping probes that recognize unique and moderately repetitive sequences along the entire length of individual chromosomes

32. Advantages – - Many more cells can be examined at a single time. - Metaphases are not essential, so abnormalities can be detected in non dividing cells. - Can be performed in a shorter period of time. - Abnormalities that cannot be detected by conventional cytogenetic analysis may be detected. Main disadvantage – - Only those abnormalities that are specifically sought will be found whereas conventional analysis permits all chromosomes to be evaluated

33. SPECTRAL KARYOTYPING (multicolor fluorescence in-situ hybridization) 24-colour, multi-chromosomal painting assay that allows visualization of all human chromosomes in one experiment. Uses – Ability to detect complex chromosomal rearrangements. Identifies marker chromosomes– makes this highly sensitive and valuable tool for identifying recurrent chromosomal abnormalities.

34. Spectral karyotyping chromosomes of a single cell Pepsin treatment( at 37 degree C for 3-5 min) labeled with a different combination of fluorescent dyes and allowed to hybridize, specific for each chromosome Imaged immediately and Spectral karyotype done using SPK View software

35. SPK

36. Comparative genomic hybridization 2 genomes Test DNA Normal DNA Labeled with 2 different fluorescence(green and red) dyes Allowed to hybridize 2 samples are equal focal deletion or Produce yellow fluorescence duplication fluorescence skewed towards green or red.

37. Uses Has higher sensitivity Can be performed using DNA extracted from fixed as well as tumor sample Technique makes it possible to perform a genome wide scan for structural alteration even on those cases for which other cytological analysis is not feasible or successful .

38. CHROMOSOMAL ABNORMALITIES NUMERICAL STRUCTURAL Karyotype with abnormal no. of chromosomes Include chromosome losses & gains Alterations in structure Include loss, rearrangements or gain of chromosome segments

39. Numerical abnormalities Haploid- gametes 23 or N Diploid- 46 or 2N Euploid - exact multiple of N eg : 3N (triploid), 4N ( tetraploid ) Aneuploid - indicates noneuploid , loss or gain of single chromosomes eg : monosomy , trisomy Most common mechanism of aneuploidy-nondisjunction of chromosomes

40. Nondisjunction in Meiosis I, results in 2 gametes with parental chromosomes that fail to separate and 2 nullisomic gametes

41. Nondisjunction in meiosis II,results in 1 gamete with two identical chromosomes, 1 nullisomic and 2 normal gametes.

42. Monosome : fertilization of nullisomic with normal gamete Trisome : fertilization of gamete retaining both paternal and maternal or both copies of either maternal or paternal with normal gamete Mosaicism : Nondisjunction when occurs in mitosis, a condition where individual has two or more cell lines of different chromosomal constitution derived from same zygote.

43. Structural abnormalities Translocation Reciprocal translocation Robertsonian translocation

44. Inversion Paracentric inversion Pericentric inversion

45. Deletion Terminal deletion Inserstitial deletion

46. Microdeletions : - Subtype of chromosome deletion that can be observed only in banded chromosomes or in some cases using molecular genetic approaches .

47. Duplications: - Intra chromosomal gain of chromatin lying in the same linear orientation (direct) reverse orientation (inverted ) with respect to centromere .

48. Insertion Interchromosome insertion Intrachromosome insertion Direct insertion Inverted insertion

49. Isochromosomes – either two identical short arms or two identical long arms. This occurs as a result of transfers split instead of longitudinal split during meiosis and mitosis.

50. Ring chromosome- these are formed when a break occurs on each arm of chromosomes followed by fusion of the exposed ends to create a circular structure. The distal fragments are lost because they lack the centromere .

51. Uniparental disomy : - A condition in which one parent has contributed 2 copies of chromosome and other parent has contributed no copies. - Ex: Prader - Willi syndrome Angelman syndrome

52. CYTOGENETIC ABNORMALITIES Chromosomal disorders Autosomes Sex chromosomes Cancer cytogenetics Soft tissue tumors Hematological disorders

53. DOWNS SYNDROME John Langdon Down in1866 Trisomy of chromosome 21 1 in 700 live births Major cause of mental retardation Maternal age has a strong influence – as the age increases the risk of down syndrome increases

54. Trisomy 21 Karyotype and FISH

55. General Hypotonia with tendency to keep mouth open Protruding tongue Craniofacial Brachycephaly with flat occiput Mild microcephaly Upslanting palpebral fissures Late closure of fontanelles Aplasia of frontal sinus Low nasal bridge Inner epicanthal folds

56. Eyes Speckling of iris ( Brushfield spots) Fine lens opacity,refractive error Nystagmus,strabismus,blocked tear duct Ears Small,overfolding of upper helix Small or absent ear lobes Hearing loss Skin loose folds in posterior neck (infancy) Cutis marmorata – extremities

57. Hands Short metacarpal and phalanges Hypoplasia of mid phalanx of 5th finger Single palmar deep flexion crease-simian crease

58. Feet Wide gap between 1st and 2nd toes rocker-bottom feet Cardiac Endocardial cushion defects-40% VSD,PDA,ASD,MVP AR by 20yrs of age

59. EDWARD SYNDROME TRISOMY 18 SYNDROME 1 per 6,000 newborn babies,<5% survive to term 47,XY/XX+18 Second most common autosomal trisomy

60. Trisomy 18 syndrome Karyotype and FISH

61. General Prenatal growth deficiency Craniofacial Characteristic facial features Small ear,small mouth, Retrognathia

62. Hands and feet clenched hand,overlapping of fingers Nail hypoplasia,short big toes rocker bottom feet Thorax short sternum,small nipples Abdominal wall unbilical hernia,small pelvis Omphalocele -protrusion of bowel into umbilical cord others VSD,cryptorchidism,hirsutism

63. PATAU SYNDROME TRISOMY 13 SYNDROME 1 in every 5,000 births 47,XY,+13 Craniofacial Mental retardation Microcephaly,microphthalmia , coloboma of iris Cleft lip,palate /both Abnormal helices,low set ears Skin Capillary hemangioma,loose skin Hands & feet Distal palmar triradii,flexion of fingers Polydactyly Cardiac VSD,PDA. Others Cryptorchidism,bicornuate uterus

64. Trisomy 13 syndrome Karyotype and FISH

65. TURNERS SYNDROME 45X SYNDROME - X0, Complete or partial monosomy of X chromosome characterized by hypogonadism in phenotypic females. Henry Turner – 1938 1 in 2000 live born females.

66. Karyotyping in Turner

67. Classic facial features Epicanthal folds Down-slanting palpebral fissures Flat nasal bridge Receding chin Lowset ears Excessive nuchal skin folds . Webbed neck

68. KLINEFELTERS (XXY) SYNDROME Male hypogonadism 2 or more X chromosomes and one or more Y chromosomes. Harry Klinefelter - 1942 Most common cause of hypogonadism and infertility 1 in 500 males affected Classic pattern – 47XXY karyotype in 82% of cases. Other mosaic patterns – 46XY/47XXY, 47XXY/48XXXY, 48XXXY/49XXXXY.

69. Karyotype 47,XXY

70. Performance Normal to low IQ Delayed speech, Poor memory Behavioral problems Problems with psychosocial adjustment Growth Long limbs, Low upper to lower segment ratio Tall and slim stature Gonads Hypogonadism, Hypogenitalism Others Elbow dysplasia,  FSH and Estradiol  Testosterone, Gynecomastia

71. XXXXX SYNDROME PENTA X SYNDROME First described by Dr.Nirmala kesaree and Wooly in 1963. Found the abnormality in prisoners in America.

72. Karyotype of XXXXX

73. Mental retardation Prenatal growth retardation Short stature Microcephaly Hypertelorism Low set ears Mongoloid slant Abnormal behavior Clinodactyly of fingers

74. CRI-DU-CHAT SYNDROME(5p-) Deletion of short arm of chromosome 5 (5p-) Critical region : 5p15 1 in 15,000 to 1 in 50,000

75. 5p-

76. Microcephaly Round face Hypertelorism Epicanthal folds Downward slanting of palpebral fissures Strabismus – often divergent Low set/poorly formed ears Facial asymmetry Cat like cry- mewing of a cat, due to abnormal laryngeal development,become less pronounced with increasing age

77. Prader-willi syndrome 1 in 15,000. Mechanism: Deletion of 15q at q11-q13(paternally derived)-75% Maternal UPD – 2 maternal,no paternal copies of 15q – 20% Chromosomal translocation involving proximal 15q – 5%

78. Craniofacial Almond shaped eyes Upslanting palpebral fissures Strabismus Thin upper lip Performance Mental retardation mild 63%,moderate 31% Excess appetite obsession with eating obesity Hands and feet Small Narrow hands,straight ulnar border Genitalia Small penis,cryptorchidism Hypoplastic labia minora & clitoris Hypogonadism

79. Angelman syndrome HAPPY PUPPET SYNDROME – abnormal puppet like gait,characteristic facies,paroxysms of laughter,due to brain stem defect – not apparently associated with happiness Mechanism Deletion of 15q11 ( maternal origin )-75% Paternal uniparental disomy in 2% Imprinting mutation 2%

80. Happy disposition, an open mouth expression, widely spaced teeth, and a pronounced mandible

81. Confirmation of the diagnosis of CML Confirmation of blast crisis of CML Diagnosis of Acute leukemias Diagnosis of lymphoproliferative disorders Diagnosis of non hodgkins lymphomas Clinical applications of cytogenetics in hematological disorders

82. Chronic Myeloid Leukaemia -(Ph+) t(9:22)(q34;q11)/BCR-ABL abnormality- Philadelphia chromosome , identified in approximately 92 % of CML patients Other abnormalities Del(9q) +8 i (17q)

83. Polycythemia vera (PV) The most common anomalies are - +8 , – 7, or a del(7q) del(11q) del(13q) del(20q ).

84. Acute Myeloid Leukaemia Broadly classified as being favorable, intermediate or poor prognostic types Eg –extensive numerical/structural karyotype abnormality aggressive myelodysplastic background t(15:17) t(8:21) favorable inv (16) or related t(16:16)

85. Acute Lymphoblastic Leukaemia most common - t(9:22 ) Tumor genetics →used for risk evaluation - hyperdiploidy (>52 chr ) → favorabble - t(12:21 ) → favorable - t(1:19 ) → unfavorable - t(9:22 )→ unfavorable - 11q23 translocation like t(4;11), t(11;19) → unfavorable - hypodiploidy (<40 chr ) →unfavorable

86. Lymphoproliferative disorders CLL O nly 50 % of CLL patients have detectabl chromosomal abnormalities Trisomy 12 – more common – worse prognosis Less often structural abnormalities seen – del(13q), del(14q), del(17p)/- 17, del(6q) Multiple myeloma structural abnormalities of chromosome- t(14;16), t(4;14)

87. Non hodgkin’s lymphoma(NHL) t(14;18) in follicular lymphoma t(8;14), t(2;8) and t(8;22) in Burkitt lymphoma t(11;14) in mantle cell lymphoma t(3;22)or t(3;14) in diffuse large B-cell lymphomas (DLBCL) t(2;5) or t(1;2) in Anaplastic large cell lymphoma (ALCL)

88. NEED OF CYTOGENETICS IN SOFT TISSUE TUMORS Understanding of soft tissue tumor biology A substantial set of soft tissue tumors contain specific karyotypic abnormalities and thus helps in diagnosis Provide insight into pathogenesis, classification, prognostic factors.

89. A Karyotype from a lipoma shows the most common rearrangement t(3;12)( q27;q15)

90. Karyotype of a Ewing’s tumor showing translocation of chromosome 11 and 22, chromosome 3 on right side is shorter than its partner because of a deletion.

91. Karyotype of a benign schwannoma with monosomy of chromosome 22

92. Complex Karyotype of a malignant peripheral nerve sheeth tumor showing aneuploid with numerous chromosomal gain, losses and rearrangement.

93. Karyotype of dermatofibrosarcoma protuberans showing supernumerary ring chromosome.

94. Conclusion

95. Bridge JA, Sandberg AA. Cytogenetics . In: Andersons pathology. 10 th ed. Mosby publications; 1996. p.223-57. Chen H. Atlas of genetic diagnosis and counselling . New jersy : Humana press publishers; 2006. Kuman V, Abbas AK, Fausto N. Genetic disorders. In: Robbins and cotran pathologic basis of disease. 8 th ed. Philadelphia: Elsevier publication; 2004 . p.145-92 . Rimoin DL, Connor JM, Pyeritz RE, Korf BR. Chromosomal disorders. In: Principles and practice of medical genetics. 4 th ed. London(UK): Churchill livingstone ; 2002. vol 1. p.1129-1202 Fletcher AJ. Cytogenetic analysis of soft tissue tumors. In: enzinger’s and weiss’s soft tissue tumors. 5 th ed. Kathleen neely , jhon canelon (USA, UK); 2008. p. 125-141 Kaushansky k, litchman AM, Beutler E, Kipps TJ, Seligsohn U, Prchal TJ. Cytogenetics and molecular abnormalities. In: wiiliams hematology 8 th ed. United states; 2010 .