Cytogenetics 2 Sioban SenGupta

2. Clinical significance of chromosome abnormalities Meiosis Numerical Structural

3. Meiosis Only in germ cells Occurs in two stages Meiosis I DNA replication 46 Chromosomes 92 Chromatids / 92 dsDNA Reduction Division separation of chromosome pairs 23 Chromosomes 46 Chromatids / 46 dsDNA Meiosis II Separation of chromatids 23 chromosomes 23 chromatids / 23 dsDNA

4. Meiosis I, Prophase I Leptotene chromosomes become apparent Zygotene homologous chromosomes pair synapsis form tetrad Pachytene crossing over occurs Diplotene chromosomes start to separate but held together by chiasmata Diakinesis Further shortening of homologous chromosomes

5. Meiosis IMetaphase I -TelophaseI Metaphase I Nuclear membrane disappears, tetrads move to equatorial position attach to spindle Anaphase I dyads of sister chromatids move to opposite poles - disjunction each pole haploid - mix of maternal and paternal Telophase I New nuclear membrane forms Secondary spermatocytes or oocytes

6. Meiosis II Prophase II nuclear membrane disappears 23 chromosomes condense Metaphase II centromeres divide and dyads move to equator Anaphase II sister chromatids move to opposite poles Telophase II new nuclear membrane forms (Spermatids or Ova) Non-disjunction failure of paired chromosomes or sister chromatids to separate at meiosis I or II

7. Synapsis Happens at prophase I – zygotene Close association of homologous chromosomes Via proteinaceous structure - synaptonemal complex Starts at telomeres and proceeds towards centromeres Each chromosome composed of two chromatids - tetrad

8. Crossing over/recombination Happens at prophase I – pachytene Tetrad Exchange homologous segments between non-sister chromatids Chromatids held together where crossed over (chiasmata)

9. Crossing over/recombination 3 proteins involved: Endonuclease makes ‘nicks’ in DNA U-protein Nicked sites targeted by U-protein unwinds 100s of bp of DNA R protein facilitates re association Nicks necessary for crossing over – repaired as cross over Preferential localisation of nicks in moderately repetitive DNA sequences so does not occur in coding regions

10. Chiasmata Site of recombination chiasmata Hold homologues together after recombination completed and chromosomes desynapsed Chiasmata ensure proper orientation of chromosomes on meiosis I spindle and thereby promote correct segregation Can see numbers of crossing overs Chromosomes 1,2 = 4 Chromosomes 3,4 = 3 More in smallest pairs than expected

11. Crossing over

12. Sex chromosomes In male, X and Y chromosome Pairing occurs between homologous segments of X and Y at tips of short arms Called pseudoautosomal region

13. Cell cycle checkpoints Integrity of genetic information maintained by cell-cycle checkpoints Checkpoint control ensures proper order of events in cell cycle

14. Meiosis check points Pachytene stage of meiotic prophase is an important control point during meiosis Pachytene Checkpoint Prevents exit from pachytene Inhibits cell cycle progression when chromosome synapsis or meiotic recombination is ongoing when defects in chromosome synapsis or recombination occur Roeder and Bailis, 2000, Lee and Amon, 2001, Roeder, 1997

15. Genetic diversity Crossing over in MI swap pieces of DNA between maternal and paternal homologous chromosomes Independent assortment at end of MI paternally and maternally derived homologues assort randomly

16. Aneuploidy caused by Non-disjunction failure of homologous chromosomes to separate in anaphase I failure of sister chromatids to separate at meiosis II Anaphase lag Chromosomal loss via micronucleus formation caused by delayed movement of chromosome/chromatid during anaphase results in daughter cell deficient of that chromosome or chromatid

17. Non-disjunction during meiosis

18. Distribution of non-disjunction

19. Gametogenesis

20. Males

22. Differences in Gametogenesis Male Puberty 60-65 days 30-500 mitoses 4 spermatids 100-200 million /ejaculate Female Early embryonic development 10-50 years 20-30 1 ovum and polar bodies 1 ovum / menstrual cycle

23. Female human embryoStages in gonad 2 months gestation – oogonia start meiosis 5 months arrest in meiosis I (diplotene/dictyotene) 6 months chromosomes held together by chiasmata By puberty 100,000 remain Maximum 300-400 mature

24. Oogenesis After diplotene – dictyotene cells in state of meiotic arrest Diplotene last until puberty After puberty – LH and FSH resumes meiosis Ovulated as at start of MII Upon fertilisation – completes meiosis

25. Aneuploidy As women age some chromosomes exhibit non-disjunction in oocytes Many theories why 13, 18, 21 associated with age 16 and X only first meiotic division associated with age Most chromosome abnormalities incompatible with life Will miscarry

26. Maternal age specific estimates of trisomy among all clinically recognisable pregnancies

27. Production line hypothesis (PLH) Henderson and Edward (1968) Germ cells committed to meiosis sequentially in fetal life Released as mature ova in sequence enter meiosis Chiasmata fewer in ova laid down late in fetal life Leads to increased number of univalents Thus aneuploid offspring in older females Evidence both supporting (Polani and Crolla, 1991) and refuting (Speed and Chandley, 1983)

28. Depleted oocyte hypothesis (DOH) Warburton (1989) as women age decreasing number of antral stage follicles per cycle thus increased likelihood of ovulating sub-optimal oocytes may include those with aberrant recombination

29. Parental origin of aneuploidy Paternal % Maternal % Trisomy 13 15 85 Trisomy 18 10 90 Trisomy 21 5 95 45,X 80 20 47,XXX 5 95 47,XXY 45 55 47,XYY 100 0

30. Down syndrome type 95% standard trisomy 1% mosaics Due to increase in maternal age 4% translocations no age effect

31. Chromosome abnormalities in humans Spermatozoa 10% Mature oocytes 25% Spontaneous miscarriage 50% Live births 0.5-1% Most due to maternal meiotic non disjunction Strongly related to maternal age Natural selection at work

32. Chromosome abnormalities in miscarriages Incidence % Trisomy 13 2 Trisomy 16 15 Trisomy 18 3 Trisomy 21 5 Other Trisomy 25 Monosomy X 20 Triploidy 15 Tetraploidy 5 Other 10

33. Chromosome abnormalities in newborns Incidence / 10,000 births Trisomy 13 2 Trisomy 18 3 Trisomy 21 15 45,X 1 47,XXX 10 47,XXY 10 47,XYY 10 Unbalanced 10 Balanced 30 Total 90

34. Triploidy Trisomy 16 Trisomy 13 &18 Trisomy 21 Klinefelters 45X rare at birth – lethal Most common in spontaneous miscarriages Completely lethal. Cause unknown 95% miscarry 80% miscarry 50% miscarry 1% at conception 98% miscarry, probably mosaic survive Chromosome abnormalities

36. Clinical significance of chromosome abnormalities Meiosis Numerical Structural Screening

37. Structural Translocations, inversions, insertions, deletions, rings What happens at meiosis? Formation of gametes that are: normal, balanced abnormal Associated with increased miscarriages Most chromosome abnormalities incompatible with life

38. Robertsonian translocation

39. 21:21 fusion At meiosis cannot form normal gametes Either disomy or nullisomy Never give normal offspring Trisomy 21 Down Monosomy 21 lethal - miscarry 6 families described 21 Down children 12 miscarriages 4 families female carrier, and 2 were male carrier

40. Reciprocal translocation 2:2 segregation Two chromosomes per gamete Could produce normal, balanced or unbalanced gametes 3:1 segregation Three chromosomes to 1 gamete One chromosome to other gamete All will be unbalanced

41. Reciprocal translocation2:2 segregation Pachytene quadrivalent Alternate gives normal or balanced gametes

42. Reciprocal translocation2:2 segregation Adjacent 1 gives unbalanced Adjacent 2 gives unbalanced

43. Reciprocal translocation3:1 segregation Pachytene quadrivalent A, C, D together – trisomy for material on C B alone – monsomy for material on B

44. Summary 2:2 Alternate A+D or B+C normal or balanced Adjacent 1 A+C or B+D unbalanced Adjacent 2 A+B or C+D unbalanced 3:1 Three A+B+C or A+B+D trisomy A+C+D or B+C+D One A or B or C or D monosomy

45. Pericentric inversion Often phenotypically normal Problems with meiosis Reverse loop forms If crossing over outside inversion no problem If crossing over within inversion loss & gain Chromosome 9 heterochromatic region Often inverted from p to q Contains repetitive non coding DNA, Frequency 1%

46. Pericentric inversion If cross over occurs within the inverted segment Normal gametes Balanced gametes inverted Unbalanced gametes Duplication of proximal end & Deletion of distal end Deletion of proximal end & Duplication of distal end

47. Paracentric inversion If cross over occurs within the inverted segment Normal gametes Balanced gametes (inverted) Acentric dicentric

48. Insertion If carrying balanced deletion/insertion OK But 50% gametes will be abnormal Could carry the deletion, insertion or both

49. Deletions Deletions are rare, as are monosomies Can be de novo or inherited due to translocation or inversion in parent Would not reproduce

50. Deletions Terminal Cri du chat, 5p15 Wolf-Hirschhorn, 4p36 Interstitial Williams, 7q11.2, microdeletion (FISH) Retinoblastoma, 13q14 Prader-Willi, 15q11.2 Angelman, 15q11.2 DiGeorge, 22q11.2

51. Cri du Chat Terminal deletion 5p15 Cries like cat Mental retardation

52. Ring chromosomes Often unstable in mitosis Often only find ring in proportion of cells Other cells usually monosomic as lack ring

53. Diagnosis of chromosome abnormalities Child born take blood and look at lymphocytes Unborn child Prenatal Diagnosis Chorionic villus sampling (CVS) Amniocentesis (AF) Fetal Blood Sampling (FBS)

54. Screening Both in first and second trimester Serum Ultrasound Indicates which should go forward for invasive procedure

55. Prenatal DiagnosisCVS Performed around 10-12 weeks Transcervical or transvaginal Aspirate or biopsy chorionic villi Examine by direct prep or culture Direct prep – cytotrophoblast display spontaneous activity – can get metaphases – result in 24 hours Culture chorionic mesoderm – for good quality G band – takes 8-14 days 1% risk miscarriage

56. CVS

57. Ambiguous resultsCVS 1% CVS get ambiguous results Could be maternal contamination More likely to see in culture than direct prep Culture artefact Usually culture several separate samples If mosaicism only in one culture probably artefact Confined placental mosaicism (CPM) True fetal mosaicism Might need to do amniocentesis/FBS to resolve problem

58. Prenatal DiagnosisAmniocentesis Most common method used Usually perform around 16 weeks 10-20 ml amniotic fluid Culture for 8-14 days Do G banding Can do a quick diagnosis on uncultured amniocytes using FISH or QF-PCR Only examine a few chromosomes 0.5-1% miscarriage rate

59. Amniocentesis

60. Ambiguous resultsAmniocentesis Usually establish 2-3 cultures 1 abnormal cell in 1 culture = artefact level 1 mosaicism or pseudo mosaicism 2 or more abnormal cells in 1 culture = could be artefact or real level 2 mosaicism, 20% chance real mosaicism 2 or more abnormal cells in 2 or more cultures = true mosaicism level 3 mosaicism To resolve need to repeat amniocentesis or do FBS

61. Molecular Cytogenetics FISH Use DNA probes for specific chromosomes Can paint metaphase Useful for quick result and identifying small areas Eg deletions, ESACs QF-PCR Quantitative fluorescent PCR Use polymorphic sites to define number of copies present Useful for quick result in prenatal diagnosis

62. Quantitative Fluorescent PCR

63. Quick result from Amniocentesis FISH Use probes for 13,21 and X, Y, 18 on two different slides takes 24 hours QF-PCR Use polymorphic markers for chromosomes 13, 18, 21 Results in 24 hours Becoming more common Can only detect abnormalities for these chromosomes Usually go on and do full karyotype - ???

64. The future Fetal cells in the maternal circulation Free fetal DNA in maternal plasma If diagnostic – no need for CVS or amniocentesis to detect chromosome abnormality