Hodgson Flashcards
What phase are cells when they are G banded?
Metaphase
How many chromosomes in humans?
46
22 autosomal pairs
1 sex pair
How are chromosomes visualised?
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
What are metacentric chromosomes?
centromere in the middle
What are Acrocentric chromosomes?
centromere at the end
What are Submetacentric chromosomes?
centromere neither at end or middle
What are Group A chromosomes?
Large metacentric
What are Group B chromosomes?
Large Submetacentric
What are Group C chromosomes?
Medium Submetacentric
What are Group D chromosomes?
Large Acrocentric
What are Group E chromosomes?
Small Submetacentric
What are Group F chromosomes?
Small Metacentric
What are Group G chromosomes?
Small Acrocentric
Where are chromosomes analysed from?
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
What is the G-Banding protocol?
- Cells are cultured to generate mitotic cells – get the cells growing
- Arrest cell cycle in metaphase (high mitotic index)
- Swell nuclei with hypotonic solution (osmosis)
- 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
- Drop fixed sample on to a glass slide
- Trypsin digest – creates pale bands – wash
- Leishmans stain – wash
- Image analysis
How does staining stain the chromosomes?
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
What is ISCN?
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
What can differences in band resolution be due to?
- Cell cycle stage – more condensed = more bands
- Tissue sample
- Experimental
How does g banding identify differences?
- 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
How is the cell cycle an issue to banding analysis?
• 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
How does tissue type alter morphology?
- 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
How does experimental technique alter analysis?
- Trypsin is a protease – cleaves peptide bonds
- Increased trypsin causes paler bands
- Overexposure causes a collapse of the chromatin – dye can’t get in
Other experimental factors which influence resolution?
- Slide aging – let the slides dry, better banding for longer
- Staining time – too long gives a lower resolution
- Chromosome spread – overlapping is hard to analyse
FISH indirect labelling
- 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 )
FISH direct labelling
- Allows for rapid diagnostic tests
- Less sensitive than indirect methods, so long probes are required
- Used in the NHS
- (nt – flourophore)
Process of metaphase and interphase FISH
• 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
What FISH techniques are used?
Chromosome Enumeration
Micro-deletion Probes
Whole Chromosome Paint
What is Chromosome Enumeration?
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
What is Micro-deletion Probes?
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
What is whole chromosome paint?
Abnormal chromosome interrogation when part of the derivative chromosome is of unknown origin
• Useful to investigate structural abnormalities involving unidentifiable chromosome regions
What are the methods of S phase synchronisation?
Deoxythymidine triphosphate (dTTP) synchronisation Fluorodeoxyuridylate (FdU) synchronization
How does dTTP synchronisation work?
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?
How is dTTP block released?
- Washing (centrifugation and subsequent suspension of lymphocytes in fresh growth media)
- 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)
How doe FdU syncronisation work?
Excess FdU inhibits dTMP synthesis
dTTP becomes rate limiting in DNA synthesis
Accumulate in S phase
Block released by addition of dTTP
What is the M phase block?
Colcemid synchronization
Blocks spindle checkpoint
How does colemid syncronise the culture?
- Inhibits tubulin polymerisation
* Synthetic analog
What are the measures of tests general reliability?
Accuracy and precision
How is accuracy defined in healthcare?
A test is accurate when the true abnormality is identified
How is precision defined in healthcare?
A test is precise when repeated analyses yield the same result, over and over again
What are the test of a likelihood of false positives and false negatives?
specificity and sensitivity
What is specificity in healthcare?
A test is specific when the false positive rate is low, that is to correctly exclude “normal” patients
What is sensitivity in healthcare?
A test is sensitive when the false negative rate is low, that is to correctly identify people who have a given disorder
How is G-banding quality assessed?
When measuring QA have to look at 4 chromosomes – need 3 out of 4 to meet criteria in both homologs
QA3, QA4, QA5, QA6
What is QA3 used for?
- Oncology only
* Example referral: Classification of haematological malignancy
What is QA4 used for?
- Exclusion of aneuploidy and large structural rearrangements
- Example referral: Prenatal diagnosis, Foetus suspected Down Syndrome
- QA4 is used in fertility
What is QA5 used for?
- 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
What is QA6 used for?
- Exclusion subtle structural rearrangements and many microdeletion syndromes
- QA6 is for blood samples of parents
- Example referral: Recurrent miscarriage (>3) family investigations
How many metaphase spreads would you look at?
10
What occurs in meiosis?
1 round of DNA replication, 2 rounds of Chromosome segregation (MI & MII)
What occurs in MI?
MI separates homologous pairs
What occurs in MII?
MII separates sister chromatids
How many miscarriges are karyotypically abnormal?
50%
90% of which have an abnormal number of chromosomes
What is the origin of most meiotic errors?
maternal in origin, due to NDJ events in MI and MII
When are NDJ events more likely?
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
What is most common abnormality?
Polyploidy then loss of sex chromosomes
What is the most common trisomy?
trisomy 16 - most are spontaneously aborted
What does trisomy 13 lead to?
Patau Syndrome
What does trisomy 18 lead to?
Edwards Syndrome
What does trisomy 21 lead to?
Down Syndrome
What are the common sex chromosome aneupoloidies?
47,XXX
47,XXY
47,XYY
Why do many sex chromosome aneuploidies come to term?
The extra X chromosome can be inactivated
The Y does not contain many genes
How are aneuplodies detected in PND?
• 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)
What results in abnormal gametes?
- MI or MII NDJ results in abnormal gametes
* Error in Meiosis is most likely by far
What is a significant risk factor to errors in meiosis?
advanced maternal age
When are errors more likely MI or MII?
MI as reduced cross overs
How is Sister chromatid cohesion mediated
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
How is Position and number of crossover events tightly regulated?
- Homologous chromosomes are covalently joined via a crossover
- Faithful disjunction therefore dependent on distal sister chromatid cohesion (relative to centromere)
Why is there a bias towars maternal NDJ
cross overs held in place till after puberty
male process is continuous
What is mosaicism?
• Two or more cell populations with different genotypes within a single individual, (developed from a single fertilized egg).
What can mosaicism affect?
the foetus, the placenta or both
• When mosaicism is found only in the placenta, this is called?
CPM – Confined Placental Mosaicism.
What is trisomy rescue?
Mitotic NDJ of the abnormal cell, which generates a normal diploid cell
how is T16 CPM caused?
trisomy rescue
What is a cause of Azoospermia and Oligospermia?
• Male gametogenesis is very sensitive to large balanced structural rearrangements and gametogenesis can be affected
What large structural rearrangements can occur?
- Reciprocal translocation (bal or unbal)
- Robertsonian translocation (unbal)
- Insertion
- Inversion
Reciprocal translocations and Robertsonian translocations are far more common than insertions and inversions
What are Robertsonian Translocations ?
They are dicentric chromosome formed from acrocentric chromosome
How many possible non-homologous and homologous robertsonian Translocations?
10 possible non-homologous and 5 homologous
In Robertsonian Translocations what do you lose?
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
ISCN for Robertsonian Translocations
First bracket is chromosome
Second bracket is breakpoint
e.g. 45,X-,t(14;21)(q10;q10)
Most common robertsonian translocations
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%
What is required for recombination?
- DSB
- Homology
- Homologous sequences need to be in the same place at the same time
How are Nucleolar Organiser Regions (NOR) involved in robertsonian translocations?
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
How are satellites involved in robertsonian translocations?
present in p11 of acrocentric ch’s
How are dicentric chromosomes made normal?
deactivation of one centromere so that only one is functional
Questions from the family?
- Are we able to have a normal child?
- Are we at risk of having further miscarriages?
- Are we at risk of having an abnormal live born child? If so, what is the risk?
- Are there any clinical interventions that can help us?
Meiosis in robertsonian translocations?
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
What is the risk of abnormal live born child if a parent has a robertsonian translocation?
10%
What woud be initiated if a person was diagnosed with a robertsonian translocation?
family studies
if noone else found would be classified as de novo
What is UPD?
Both homologous chromosomes from a parent.
What is UPID?
Two copies of the same chromosome from one parent
WHat are the 5 clinically relevant imprinted chromosomes?
6, 7, 11, 14, 15
What is imprinting at chromosome 6 associated with?
PAT Transient neonatal diabetes mellitus (DMTN
What is imprinting at chromosome 7 associated with?
MAT Russell Silver syndrome
What is imprinting at chromosome 11 associated with?
PAT Beckwith-Wiedermann syndrome
What is imprinting at chromosome 14 associated with?
MAT Temple syndrome
PAT Kagami-Ogata syndrome
What is imprinting at chromosome 15 associated with?
MAT Prader-Willi syndrome
Angelman syndrome
Mechanisms for UPD/UPID include:
- Trisomy rescue (can result in UPD)
- Monosomy rescue (results in UPID)
- Gamete Complementation (could result in either UPD or UPID)
loss of heterozygosity (LOH)
Whay are • Robertsonian translocation carriers at increased risk of UPD affected pregnancies?
predisposed to aneuploid gamete formation
Large structural rearrangements can result in:
– 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
Why do paternal carriers not usually pass on a rearrangement?
They are infertile
What are the 2 broad subtypes of band translocation?
- Recurrent – same translocation in many unrelated individuals
- Familial (unique) – see in different individuals but only if they are related
What is the most common recurrent non-Robertsonian translocation in humans?
t(11;22)(q23;q11)
What are • Translocation breakpoints characterised by?
palindromic AT-rich repeats(PATRR)
What are the clinical symptms of t(11;22)(q23;q11)?
o Male infertility
o Recurrent miscarriage
o Risk of a specific genetic disease “Emanuel Syndrome”
What is emmanuel syndrome caused by?
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)
How are carriers of emmanuel syndrome diagnosed?
when a child is diagnosed as having the disease
What are the results of MI if a parent is a carrier for emmanuel syndrome?
- Alternate segregation: alternate centromere segregate together
Results in two normal offspring
Also results in two carriers of the balanced translocation - 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 - Adjacent 2 segregation: Homologous chromosomes segregate to the same pole- Adjacent centromeres segregate together
Results in 4 unbalanced chromosomes that result in spontaneous abortions - Nondisjunction:
Chromatids segregate in a 3:1 manner
4 unbalanced gametes
When is the 11;22 translocation formed de novo?
in spermatogenesis
How is the 11;22 translocation likely to be formed?
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
