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Human reproduction and fertility > Hodgson > Flashcards

Hodgson Flashcards

1
Q

What phase are cells when they are G banded?

A

Metaphase

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2
Q

How many chromosomes in humans?

A

46
22 autosomal pairs
1 sex pair

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3
Q

How are chromosomes visualised?

A

Cultured human cells (need dividing cells)
Extract nuclei and fix to a microscope slide
Partial digestion of chromatin (Trypsin)
Followed by staining (Leishmans)
Microscopic analysis

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4
Q

What are metacentric chromosomes?

A

centromere in the middle

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5
Q

What are Acrocentric chromosomes?

A

centromere at the end

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6
Q

What are Submetacentric chromosomes?

A

centromere neither at end or middle

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7
Q

What are Group A chromosomes?

A

Large metacentric

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8
Q

What are Group B chromosomes?

A

Large Submetacentric

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9
Q

What are Group C chromosomes?

A

Medium Submetacentric

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10
Q

What are Group D chromosomes?

A

Large Acrocentric

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11
Q

What are Group E chromosomes?

A

Small Submetacentric

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12
Q

What are Group F chromosomes?

A

Small Metacentric

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13
Q

What are Group G chromosomes?

A

Small Acrocentric

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14
Q

Where are chromosomes analysed from?

A

o Blood samples from mother and father - looking at inherited defects
o Samples are foetal epithelial cells from amniotic fluid
o Sometimes placental material
o Most samples are not actively growing and dividing (cancer cells are the exception

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15
Q

What is the G-Banding protocol?

A
  1. Cells are cultured to generate mitotic cells – get the cells growing
  2. Arrest cell cycle in metaphase (high mitotic index)
  3. Swell nuclei with hypotonic solution (osmosis)
  4. Kill cells using fixative (3:1 Methanol:Acetic Acid) – spreads samples out and also stops samples form condensing and reduce risk of staff becoming infected
  5. Drop fixed sample on to a glass slide
  6. Trypsin digest – creates pale bands – wash
  7. Leishmans stain – wash
  8. Image analysis
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16
Q

How does staining stain the chromosomes?

A

Dark bands: AT rich
Light bands: GC rich
Open chromatin is stained more – dark, better access to binding pockets
Staining is highly reproducible between samples of the same patient and between patients

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17
Q

What is ISCN?

A

international system for cytogenetic nomenclature
Centromere is p10 or q10
Numbering increases away from the centromere
Q is the longer arm
P is the shorter arm

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18
Q

What can differences in band resolution be due to?

A
  1. Cell cycle stage – more condensed = more bands
  2. Tissue sample
  3. Experimental
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19
Q

How does g banding identify differences?

A
  • Looking at differences between the banding on two homologs and for that to be liked to a clinical phenotype
  • Differences can be genetic
  • Subjective and analysis is dependent on competency
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20
Q

How is the cell cycle an issue to banding analysis?

A

• Asynchronous cultures:
o Contains dividing cells at all stages of mitosis, and G0 (quiescence)
o Want longer chromosomes so later through metaphase
o These will however be more overlapping

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21
Q

How does tissue type alter morphology?

A
  • Blood samples give much longer chromosomes
  • Foetal material is shorter
  • Stem cells are even shorter – not fertility related
  • Resolution may correlate to state of differentiation – more differentiated = more resolution
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22
Q

How does experimental technique alter analysis?

A
  • Trypsin is a protease – cleaves peptide bonds
  • Increased trypsin causes paler bands
  • Overexposure causes a collapse of the chromatin – dye can’t get in
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23
Q

Other experimental factors which influence resolution?

A
  1. Slide aging – let the slides dry, better banding for longer
  2. Staining time – too long gives a lower resolution
  3. Chromosome spread – overlapping is hard to analyse
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24
Q

FISH indirect labelling

A
  • Potential for greater sensitivity than direct, but slower
  • More labour intensive
  • Add probe then fluorophore
  • Not used clinically – is commonly used in research labs
  • (nt – hepatin – fluorophore )
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25
Q

FISH direct labelling

A
  • Allows for rapid diagnostic tests
  • Less sensitive than indirect methods, so long probes are required
  • Used in the NHS
  • (nt – flourophore)
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26
Q

Process of metaphase and interphase FISH

A

• Make DNA single stranded
o heat sample to 75-78 degrees
o denature DNA a bit but not destroy structures
• Anneal probe
o 37 to 40 degrees
• Many wash steps to remove probes bound to non-complementary regions
• DAPI – used as a counter stain for both methods

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27
Q

What FISH techniques are used?

A

Chromosome Enumeration
Micro-deletion Probes
Whole Chromosome Paint

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28
Q

What is Chromosome Enumeration?

A

For common aneuploidies (X turners, Y aggression, 21 downs, 18 Edwards, 13 Patau)
• Probes tend to be very large, sometimes 100s of kb.
• Bright signal allowing rapid hybridisation times
• Bright signals allow for a rapid and less ambiguous analysis
• Probes are specific to the alpha satellite DNA sequences at the loci indicated above

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29
Q

What is Micro-deletion Probes?

A

Usually unbalanced foetal karyotypes due to “abnormal” inheritance of chromosomes from a parent with a balanced rearrangement
• Diagnostic test for Cri du Chat and SOTOS
• Often multiple diagnostic probes are combined, to save money.
• One probe is used as the +ve control for the other

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30
Q

What is whole chromosome paint?

A

Abnormal chromosome interrogation when part of the derivative chromosome is of unknown origin
• Useful to investigate structural abnormalities involving unidentifiable chromosome regions

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31
Q

What are the methods of S phase synchronisation?

A 
Deoxythymidine triphosphate (dTTP) synchronisation
Fluorodeoxyuridylate (FdU) synchronization
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32
Q

How does dTTP synchronisation work?

A

Excess dTTP inhibits the reduction of CDP by the enzyme Ribonucleotide Reductase
dCTP becomes rate limiting in DNA synthesis
Stay in S phase until relased?

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33
Q

How is dTTP block released?

A
  1. Washing (centrifugation and subsequent suspension of lymphocytes in fresh growth media)
  2. Addition of dCTP, bypassing the need for Ribonucleotide Reductase (preferred in healthcare as it is better for health and safety as centrifugation can break tubes and put staff at risk of infection)
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34
Q

How doe FdU syncronisation work?

A

Excess FdU inhibits dTMP synthesis
dTTP becomes rate limiting in DNA synthesis
Accumulate in S phase
Block released by addition of dTTP

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35
Q

What is the M phase block?

A

Colcemid synchronization

Blocks spindle checkpoint

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36
Q

How does colemid syncronise the culture?

A
  • Inhibits tubulin polymerisation

* Synthetic analog

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37
Q

What are the measures of tests general reliability?

A

Accuracy and precision

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38
Q

How is accuracy defined in healthcare?

A

A test is accurate when the true abnormality is identified

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39
Q

How is precision defined in healthcare?

A

A test is precise when repeated analyses yield the same result, over and over again

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40
Q

What are the test of a likelihood of false positives and false negatives?

A

specificity and sensitivity

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41
Q

What is specificity in healthcare?

A

A test is specific when the false positive rate is low, that is to correctly exclude “normal” patients

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42
Q

What is sensitivity in healthcare?

A

A test is sensitive when the false negative rate is low, that is to correctly identify people who have a given disorder

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43
Q

How is G-banding quality assessed?

A

When measuring QA have to look at 4 chromosomes – need 3 out of 4 to meet criteria in both homologs
QA3, QA4, QA5, QA6

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44
Q

What is QA3 used for?

A
  • Oncology only

* Example referral: Classification of haematological malignancy

45
Q

What is QA4 used for?

A
  • Exclusion of aneuploidy and large structural rearrangements
  • Example referral: Prenatal diagnosis, Foetus suspected Down Syndrome
  • QA4 is used in fertility
46
Q

What is QA5 used for?

A
  • Exclusion of aneuploidy and large and more subtle structural rearrangements
  • Example referral: Prenatal diagnosis, Ultrasound Scan (16-20w gestation) morphological abnormalities detected
  • First scan dates a pregnancy, second scan looks for abnormalities – referral after 2nd scan
47
Q

What is QA6 used for?

A
  • Exclusion subtle structural rearrangements and many microdeletion syndromes
  • QA6 is for blood samples of parents
  • Example referral: Recurrent miscarriage (>3) family investigations
48
Q

How many metaphase spreads would you look at?

A

10

49
Q

What occurs in meiosis?

A

1 round of DNA replication, 2 rounds of Chromosome segregation (MI & MII)

50
Q

What occurs in MI?

A

MI separates homologous pairs

51
Q

What occurs in MII?

A

MII separates sister chromatids

52
Q

How many miscarriges are karyotypically abnormal?

A

50%

90% of which have an abnormal number of chromosomes

53
Q

What is the origin of most meiotic errors?

A

maternal in origin, due to NDJ events in MI and MII

54
Q

When are NDJ events more likely?

A

when there are fewer crossovers between homologous chromosomes, and when only a single crossover homologous chromosomes
NDJ is more likely when positioned distal to the centromere

55
Q

What is most common abnormality?

A

Polyploidy then loss of sex chromosomes

56
Q

What is the most common trisomy?

A

trisomy 16 - most are spontaneously aborted

57
Q

What does trisomy 13 lead to?

A

Patau Syndrome

58
Q

What does trisomy 18 lead to?

A

Edwards Syndrome

59
Q

What does trisomy 21 lead to?

A

Down Syndrome

60
Q

What are the common sex chromosome aneupoloidies?

A

47,XXX
47,XXY
47,XYY

61
Q

Why do many sex chromosome aneuploidies come to term?

A

The extra X chromosome can be inactivated

The Y does not contain many genes

62
Q

How are aneuplodies detected in PND?

A

• Rapid service (FISH or Cell-free foetal DNA QPCR) – don’t need to grow cells so are quicker
o Can send preliminary report within 24 hours
• Karyotype Analysis (Gold standard)

63
Q

What results in abnormal gametes?

A
  • MI or MII NDJ results in abnormal gametes

* Error in Meiosis is most likely by far

64
Q

What is a significant risk factor to errors in meiosis?

A

advanced maternal age

65
Q

When are errors more likely MI or MII?

A

MI as reduced cross overs

66
Q

How is Sister chromatid cohesion mediated

A

After DNA replication, cohesion (ring like complex) is laid down behind polymerase
Cohesion is a clamping protein of a heterodimer Smc1/Smc3
Ring is closed by:
• Mitosis – Scc1
• Meiosis – Rec8
Topoisomerase creates DSBs and homologous recombination repairs them

67
Q

How is Position and number of crossover events tightly regulated?

A
  1. Homologous chromosomes are covalently joined via a crossover
  2. Faithful disjunction therefore dependent on distal sister chromatid cohesion (relative to centromere)
68
Q

Why is there a bias towars maternal NDJ

A

cross overs held in place till after puberty

male process is continuous

69
Q

What is mosaicism?

A

• Two or more cell populations with different genotypes within a single individual, (developed from a single fertilized egg).

70
Q

What can mosaicism affect?

A

the foetus, the placenta or both

71
Q

• When mosaicism is found only in the placenta, this is called?

A

CPM – Confined Placental Mosaicism.

72
Q

What is trisomy rescue?

A

Mitotic NDJ of the abnormal cell, which generates a normal diploid cell

73
Q

how is T16 CPM caused?

A

trisomy rescue

74
Q

What is a cause of Azoospermia and Oligospermia?

A

• Male gametogenesis is very sensitive to large balanced structural rearrangements and gametogenesis can be affected

75
Q

What large structural rearrangements can occur?

A
  • Reciprocal translocation (bal or unbal)
  • Robertsonian translocation (unbal)
  • Insertion
  • Inversion

Reciprocal translocations and Robertsonian translocations are far more common than insertions and inversions

76
Q

What are Robertsonian Translocations ?

A

They are dicentric chromosome formed from acrocentric chromosome

77
Q

How many possible non-homologous and homologous robertsonian Translocations?

A

10 possible non-homologous and 5 homologous

78
Q

In Robertsonian Translocations what do you lose?

A

Lose a small piece of the chromosome (p arm), contains:
• Some genes present in many copies – tolerable
• Repetitive DNA
• Microsatellites
Not linked to adverse phenotype

79
Q

ISCN for Robertsonian Translocations

A

First bracket is chromosome
Second bracket is breakpoint
e.g. 45,X-,t(14;21)(q10;q10)

80
Q

Most common robertsonian translocations

A

45,X-,t(13;14)(q10;q10) - 76%
45,X-,t(14;21)(q10;q10) – 10%
The rest are equally common an account for the remaining 14%

81
Q

What is required for recombination?

A
  • DSB
  • Homology
  • Homologous sequences need to be in the same place at the same time
82
Q

How are Nucleolar Organiser Regions (NOR) involved in robertsonian translocations?

A

Ribosomal RNA Genes rDNA are found at p12 of each of the 5 acrocentric chromosomes
Nucleolus is the site of RNA biogenesis in G1
Model predicts loss of rDNA genes of which there is no known adverse clinical effects

83
Q

How are satellites involved in robertsonian translocations?

A

present in p11 of acrocentric ch’s

84
Q

How are dicentric chromosomes made normal?

A

deactivation of one centromere so that only one is functional

85
Q

Questions from the family?

A
  1. Are we able to have a normal child?
  2. Are we at risk of having further miscarriages?
  3. Are we at risk of having an abnormal live born child? If so, what is the risk?
  4. Are there any clinical interventions that can help us?
86
Q

Meiosis in robertsonian translocations?

A 
Three scenarios:
1.	4 gametes – all balanced: 
2x normal gametes (normal) 
2x  (“normal” but carrier)
2.	4 gametes – all unbalanced: 
2x disomic 
2x nullisomic 
3.	4 gametes – all unbalanced: 
2x disomic 
2x nullisomic
87
Q

What is the risk of abnormal live born child if a parent has a robertsonian translocation?

A

10%

88
Q

What woud be initiated if a person was diagnosed with a robertsonian translocation?

A

family studies

if noone else found would be classified as de novo

89
Q

What is UPD?

A

Both homologous chromosomes from a parent.

90
Q

What is UPID?

A

Two copies of the same chromosome from one parent

91
Q

WHat are the 5 clinically relevant imprinted chromosomes?

A

6, 7, 11, 14, 15

92
Q

What is imprinting at chromosome 6 associated with?

A

PAT Transient neonatal diabetes mellitus (DMTN

93
Q

What is imprinting at chromosome 7 associated with?

A

MAT Russell Silver syndrome

94
Q

What is imprinting at chromosome 11 associated with?

A

PAT Beckwith-Wiedermann syndrome

95
Q

What is imprinting at chromosome 14 associated with?

A

MAT Temple syndrome

PAT Kagami-Ogata syndrome

96
Q

What is imprinting at chromosome 15 associated with?

A

MAT Prader-Willi syndrome

Angelman syndrome

97
Q

Mechanisms for UPD/UPID include:

A
  1. Trisomy rescue (can result in UPD)
  2. Monosomy rescue (results in UPID)
  3. Gamete Complementation (could result in either UPD or UPID)
    loss of heterozygosity (LOH)
98
Q

Whay are • Robertsonian translocation carriers at increased risk of UPD affected pregnancies?

A

predisposed to aneuploid gamete formation

99
Q

Large structural rearrangements can result in:

A

– Loss of or reduced fertility in males due to a failure of spermatogenesis.
– Recurrent miscarriages (unbalanced constitutional karyotype of the foetus)
– Live born abnormal child

100
Q

Why do paternal carriers not usually pass on a rearrangement?

A

They are infertile

101
Q

What are the 2 broad subtypes of band translocation?

A
  • Recurrent – same translocation in many unrelated individuals
  • Familial (unique) – see in different individuals but only if they are related
102
Q

What is the most common recurrent non-Robertsonian translocation in humans?

A

t(11;22)(q23;q11)

103
Q

What are • Translocation breakpoints characterised by?

A

palindromic AT-rich repeats(PATRR)

104
Q

What are the clinical symptms of t(11;22)(q23;q11)?

A

o Male infertility
o Recurrent miscarriage
o Risk of a specific genetic disease “Emanuel Syndrome”

105
Q

What is emmanuel syndrome caused by?

A

3:1 malsegregation of the abnormal Ch22 and the supernumerary inheritance of this derivative chromosome.
A child with Emanuel syndrome is:
47,XY,+der(22)t(11;22)(q23;q11)

106
Q

How are carriers of emmanuel syndrome diagnosed?

A

when a child is diagnosed as having the disease

107
Q

What are the results of MI if a parent is a carrier for emmanuel syndrome?

A
  1. Alternate segregation: alternate centromere segregate together
    Results in two normal offspring
    Also results in two carriers of the balanced translocation
  2. Adjacent 1 segregation: Non-homologous chromosomes segregate to the same pole – adjacent centromere segregate together
    All gametes are unbalanced – all would give an unbalanced zygote that would spontaneously abort
  3. Adjacent 2 segregation: Homologous chromosomes segregate to the same pole- Adjacent centromeres segregate together
    Results in 4 unbalanced chromosomes that result in spontaneous abortions
  4. Nondisjunction:
    Chromatids segregate in a 3:1 manner
    4 unbalanced gametes
108
Q

When is the 11;22 translocation formed de novo?

A

in spermatogenesis

109
Q

How is the 11;22 translocation likely to be formed?

A

involve the colocalisation of PATRR11 and PATRR22 during late spermatogenesis, DSB formation and subsequent aberrant repair.
DSB repair is likely to occur via NHEJ, as late spermatids cannot undergo HR

Human reproduction and fertility (4 decks)

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