hodgson Flashcards
what is the g banding protocol
- cells cultured to generate mitotic cells
- arrest cell cycle in metaphase
- swell nuclei with hypotonic solution
- kill cells using fixative
- drop fixed sample onto glass slide
- trypsin digest
- leishmans stain
- image analysis
why do bands appear dark and light
dark bands are AT rich - open chromatin
pale bands are GC rich - closed chromatin
what does fixative do
block chromosome condensation, kill everything in sample eg removes infectious disease
what can the centromere be referred to as
p10 or q10
which type of fish is not used clinically in the UK
indirect label FISH
what does trisomy 18 lead to
edwards syndrom
wha does trisomy 13 lead to
pateu syndrome
what does a small deletion of 5p lead to
cri du chat
what does banding resolution depend on
- cell cycle stage
- tissue sample
- experimental (slide aging, staining time, chromosome spread)
what are the steps to direct labelling FISH
- take microscope slide with DNA
- make target DNA single stranded by heating sample to 75-78 degrees
- Anneal probe at 37-40 degrees
- series of washes to remove unbound probes
- DAPI used as a counter stain
what are the 3 main types of FISH probe
- chromsome enumeration probes for common aneuploidies
- microdeletion probes
- whole chromosome pait
when would you use whole chromosome paint
when youre unsure of origin of a chromsome
At high concentrations what inhibits ribinucleotide reductase which converts CDP to dCDP meaning the concs of dCTP become rate limiting and the lymphocytes remain in S phase
dTTP
how can we cause S phase synchronisation
high levels of dTTP
how is the thymidine block released
washing (centrifugation)
- addition of dCTP
which is the preferred way of releasing the thymidine block
addition of CTP to bypass the need for ribonucleotide reductase (what excess dTTP is limiting)
why is addition of CTP the preferred method of overcoming the thymidine block
health and safety risk to centrifugation - can fracture a tube and a sample may be contaminated with infectious disease
what is another way of synchronising cells in S phase
addition of Fdu
how does addition of Fdu cause cells to be synchronised in S phase
excess Fdu blocks the synthesis of dTMP which is a precursor of dTTP so reduces its availability for DNA synthesis
how can the Fdu block be released
addition of excess dTTP but addition of too much can block the cycle again by inhibiting ribonucleotide reductase
what is colcemid
microtubule inhibitor that arrests cells in metaphase
how does colcemid inhibit microtubules
binds to soluble tubulin, colcemid-tubulin complex may still polarise but with significantly reduced efficiency
microtubule stability is reduced, preventing spindle formation
what are the 4 main sections of quality
accuracy, precision, specificity and sensitivity
what do we mean by accuracy
a test is accurate when the true abnormality is identified
what do we mean by precision
a test is precise when repeated analyses yield the same result over and over again
when is a test specific
when false positive rate is low
when is a test sensitive
when false negative rate is low
what QA is used in fertility services
QA4
what is much more susceptible to structural arrangements
spermatogenesis - oogenesis is much more robust
rDNA encoded on p arm of acrocentric chromosomes 13,14,15,21,22. Repetitive DNA can be lost or gained with no abnormal clinical phenotype T/F
T
what are the 3 different strategies for cell culture synchronisation
dTTP, FdU (s phase)
colcemid (metaphase)
the higher the QA number the higher the…
resolution
what is required to meet a QA
3/4 chromsomes with specified banding seen in both homologs
what is QA4 used for
Exclusion of aneuploidy and large structural
rearrangements
what is QA5 used for
Exclusion of aneuploidy and large and more
subtle structural rearrangements
what is QA6 used for
Exclusion subtle structural rearrangements and many microdele1on syndromes (recurrent miscarriage)
in meiosis I chromosomes pair up and are held together in what
bivalents
why is crossing over important in meiosis
holds chromosomes together
what does the first meiotic division do
separates homologous chromosomes to opposite ends of cells
what happens in M2
sister chromatids are segregated
what percentage of miscarriages are karyotypically abnormal
50%
of the karyotypically abnormal abortions what percentage had inherited wrong number of chromosomes
90%
what is inheritance of the wrong number of chromosomes largely due to
fusion of one normal gamete and one abnormal gamete and therefore errors in meiotic chromosome segregation
most meiotic errors are …. in origin due to non disjunction events in MI and MII
maternal
when are non disjunction events more likely
when there are fewer crossovers between homologous chromosomes
when there is only one crossover between homologous chromsomes when is NDJ events more likely
when crossover positioned distal to centromere (close to it)
what are the most common type of abnormalities
triploidy, loss of sex chromosomes
what is the most common trisomy at the point of conception
trisomy 16 - leads to spontaneous abortion always
what is the exception of when trisomy 16 can lead to foetus coming to term
when its confined to placenta (confined placental mosaicism)
what percentage of conceptions with trisomy 18 are expected to spontaneously abort
95%
what percentage of conceptions with trisomy 21 are expected to spontaneously abort
78%
How are these common aneuploidies
detected in a PND Service
rapid service (FISH or cell-free foetal DNA QPCR) karyotype analysis (gold standard)
After DNA replication cohesion is laid down behind the polymerase
Cohesion is a heterodimer (Smc1 and Smc3) and the ring is closed by another protein – in mitosis its Scc1 and in meiosis its
…
Rec8
early in meiosis (MI) what gets loaded onto DNA to create double stranded breaks
Spo11
the breaks created by Spo11 are then repaired by what later in MI
homologous recombination machinery (can crossover or not)
there is some inhibition in local region to prevent breaks in local region being repaired to form crossover - why
prevents too many crossovers because this is likely to non disjoin chromsomes in meiosis II
the only cohesiont that contributes to holding homologs together is positioned where
between the crossover and the telomere
the further down a cross over occurs….
the less cohesion holding them together
why is there a bias toward maternal NDJ
chromsomes are held in crossovers for years and complexes can suffer damage
the closer the crossover is to telomere the fewer cohesion holding complex together and if some are damaged means its more likely for NDJ
what does mosaicism normally result from
mitotic chromosome nondisjucntion event resulting from trisomic and monosomic cells
when mosaicism is only found in the placenta this is called what
confined placental mosaicism
- In cases of T16 CPM nearly all are caused by a trisomic conception resulting from maternal M1 non-disjunction. This means that CPM for T16 is almost always formed by what
trisomy rescue
the confinement of the trisomic cell to the placenta depends on what
when the rescue event occured and in which cell
what must cases of CPM T16 also be considered for
uniparental disomy as it is an imprinted chromsome
Trisomy 16 cells must be completely or at least predominantly confined to the placenta for a mosaic trisomy 16 conceptus to survive T/F
T
UPD(16)mat is likely to have a subtle phenotypic effect based on statistical analysis of 83 cases and therefore should be considered one of the pathogenic mechanisms of trisomy 16 during pregnancy T/F
T
Mechanisms that may lead to UPD include trisomy rescue through loss of a chromosome and what
monosomy rescue through duplication of a chromsome
what sample stops the confusion over CPM
amniotic fluid sample - know your looking at foetus tissue only
what are the most common structural rearrangement
robertsonian translocation
what do robertsonian translocations result in
the slight loss of genetic material
robertsonian translocation are dicentric chromosomes. what does this mean
contain 2 copies of centromere
what chromosomes are involved in robertsonian translocations
acrocentric chromosomes (13,14,15,21,22)
how many possible different non homologous robertsonian translocations are there
10
there are 5 possible homologous robertsonian translocations. what happens in a homologous robertsonian translocation
q arm is fused to another of the same q arm (eg 13q fuses to the other 13q)
robertsonian translocations for which chromosomes are far more common than other chromosomes
13, 14, 21
the majority of breakpoints in Robertsonian translocations are
proximal to the … region (with respect to the centromere) and therefore result in the deletion of the … region
NOR
the sequence of p arm acrocentric chromosomes at p12 are the site of rDNA genes that encode what
ribosomal RNA
- During formation of nucleolus all p arms of acrocentric chromosomes come together at one time
- You have homology in rDNA genes
- Site of greater homology is on band 11.2
T/F
T
In comparison to the less common types of Robertsonian
translocations, the breakpoints in rob(13q14q) and rob(14q21q)
were extremely consistent in location.. where did the breakpoints occr
between
pTRI-6 sequences and rDNA on chromosome 13 and between
pTRS-47 and pTRS-63 sequences on chromosome 14
the orientation of microsatellites is important - why
they are inverted so that they pair up and exchange to form dicentric chromosomes
what are the questions from a family that are normally asked for a cytogenetics investigation into recurrent miscarriage
- 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 to help
in a robertsonian transloaction how many centromeres are there between the 2 chromosomes involved
1
if the mother is the carrier of a robertsonian translocation between chromosome 12 and 14 and the father is karyotypically normal what are the chances of normal conception live born
4/12
but 50% of these will carry t(14;21)
if its male (50%) will be infertile
if the mother is the carrier of a robertsonian translocation between chromosome 12 and 14 and the father is karyotypically normal what are the chances of abnormal conception
2/12 viable live born trisomy 21
if the mother is the carrier of a robertsonian translocation between chromosome 12 and 14 and the father is karyotypically normal what are the chances of abnormal conception with spontanous abortion
6/12
do not know the exact risk of robertsonian translocations. what other factors do you have to account for
- Can get non disjunction of other chromosomes – need to account in risk
- Could get trisomy foetus by mitotic nondisjunction event
- Can have mitotic nondisjunction rescue events to correct genomic imbalance
- Trisomic conception for 14 followed by rescue event could lead to imprinting disease
what is UPD
both homologous chromosomes have been inherited from a single parent with no contribution from the other parent
how can UPD result in abnormal phenotype
imprinted genes
loss of heterozygosity resulting in recessive disease
what are three mechanisms for UPD
- trisomy rescue
- monosomy rescue (UPID)
- gamete complementation
robertsonian translocation carriers are at an increased risk of UPD affected pregnancies T/F
T
which errors are more common MI or MII
MI
when can you have a foetus with abnormality from 2 normal gametes due to…
mitotic non disjunction errors that occur after fertilisation
what an large structural rearrangements can result in (3)
reduced or loss of fertility in males due to failures in spermatogenesis
recurrent miscarriages
live born abnormal child
- Progeny tend to inherit translocations and other balanced rearrangements from their…
Mother
if mum and dad are normal how can a child carry a balanced structural rearrangement
A de novo event. in the vast majority of cases it has been inherited because the male is mosaic. majority of balanced structural arrangements happen in latter stages of spermatogenesis. balances translocations occur de novo in haploid cells that are generated
what is meant by a recurrent balanced reciprocal translocation
same translocation is seen in many different unrelated individuals
what is meant by a familial (unique) recurrent balanced translocation
see in different individuals but only if they’re related
what are 2 examples of familial balanced translocations
t(11;14)(q13;q32)
t(14;16)(q32;q23)
what is the most common recurrent non robertsonian translocation
t(11;22)(q23;q11) - approx 1 in 1000
the translocation breakpoints in t(11;22)(q23;q11) are characterised by what
palindromic AT rich repeats
what are the known clinical symptoms of t(11;22)(q23;q11)
all relate to fertility
- male infertility
- recurrent miscarriage
- risk of a specific genetic disease
t(11;22)(q23;q11) gives rise to a risk of which disease
Emanuel syndrome
what is emanuel syndrome caused by
3:1 malsegregation of the abnormal chromosome 22 and the supernumerary inheritance of this derivative chromosome
what are the clinical features of emanuel syndrome
severe mental retardation and distinctive morphological features
kidney and heart problems
what are the most common cause of spermatogenic failure
Deletions of the AZFc (azoospermia factor c) region of the Y chromosome
deletion of any one of three Yq regions (AZFa, AZFb or AZFc)
severely diminishes or extinguishes sperm production T/F
T
in AZFc deletions the region in which the proximal breakponts cluster is strikingly similar in sequence to region where the distal breakpoints cluster. homologous recombination between which two amplicons is a frequent cause of spermatogenic failure
b2 and b4
how are AZFc deletions caused
intrachromosomal homologous recombination between repeated sequence blocks called “amplicons” organised into palindromic structures showing a nearly identical sequence
the likelihood of illegitimate recombination event seems to be dependent on what 2 factors
- length of homology in repetitive sequence
- distance between repetitive sequence
(more likely to suffer when repetitive seq are longer and spacer DNA between them is smaller)
children with emanuel disease have what karyotype
47,XY,der(22)t(11;22)(q23;q11)
how can emanuel syndrome occur
Carriers of t(11;22)(q23;q11) can go through 3:1 malsegregation (NDJ) resulting in Emanuel syndrome
emanuel syndrome is caused by the malsegregation of chromosomes during meiosis I
T
what does alternate segregation where alternate centromeres segregate together lead to:
4 gametes with no genomic imbalance
2 normal gametes
2 gametes with balanced translocation
what do you get in adjacent I segregation where adjacent centromere (non-homologous) segregate together
all gametes unbalanced
Most likely outcome if these gametes were to fuse with normal male gamete would be an unbalanced zygote and most likely spontaneous abortion
what do you get if you get adjacent II segregation and adjacent centromeres (homologous) segregate together
4 largely unbalanced gametes
spontaneous abortion
what does MI nondisjunction (3:1 malsegregation) lead to
2 gametes with supernumery
emanuel syndrome
repetitive regions seem to be involved with the 11;22 translocation T/F
T
PATRR are thought to be susceptible to mis-pairing leadin to the formation of secondary structures such as stem loops which are able to induce genomic instability
T
he de-novo generation of the 11;22 translocation also appears to be restricted to which process
spermatogenesis
- The exact mechanism by which the 11;22 translocation is formed is still not fully understood, but it is likely to involve the colocalisation of PATRR11 and PATRR22 during late spermatogenesis, DSB formation and subsequent aberrant repair T/F
T
-DSB repair is likely to occur via NHEJ, as late spermatids cannot undergo what
homologous recombination
- Balanced reciprocal translocations are quite rare, but they are the most common structural rearrangement after what
robertsonian translocations
