Cytogenetics II

1
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
Oogonium
Spermatogonium
Primary oocyte
Primary spermatocyte
Secondary oocyte
Secondary spermatocytes
Polar Body I
4 spermatids
Polar Body II
Fertilized Ovum
4 spermatozoa
20
Males
Oogonium
Spermatogonium
Throughout Life
Mitosis
Primary oocyte
At Puberty
Primary spermatocyte
Meiosis Complete in 64days
Secondary oocyte
Secondary spermatocytes
Polar Body I
4 Spermatids
Polar Body II
Fertilized Ovum
4 spermatozoa
21
Oogonium
Mitosis
Primary Oocyte
Females
Secondary Oocyte Polar Body I
Fertilized Ovum Polar body II
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
Hassold et al., 1985
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
Chromosome abnormalities

• 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

35
Cytogenetics 3 Sioban SenGupta
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
2121 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

• 22 segregation
• Two chromosomes per gamete
• Could produce normal, balanced or unbalanced
  gametes
• 31 segregation
• Three chromosomes to 1 gamete
• One chromosome to other gamete
• All will be unbalanced

41
Reciprocal translocation22 segregation

• Pachytene quadrivalent
• Alternate
• gives normal or balanced gametes

42
Reciprocal translocation22 segregation

• Adjacent 1 gives unbalanced
• Adjacent 2 gives unbalanced

43
Reciprocal translocation31 segregation

• Pachytene quadrivalent
• A, C, D together trisomy for material on C
• B alone monsomy for material on B

44
Summary

• 22
• Alternate AD or BC normal or balanced
• Adjacent 1 AC or BD unbalanced
• Adjacent 2 AB or CD unbalanced
• 31
• Three ABC or ABD trisomy
• ACD or BCD
• 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
Trisomy detection in prenatal samples
Hypervariable region on chromosome 21 amplified
by F-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