Adam - G-banding, FISH and workshop

Question 1

if you identify Robertsonian translocation causing DS in foetus (or any risk really) what further investigation must you do?

Answer 1

assuming you’ve done immediate FISH, then karyotyping…

determine whether or not the mutation is de novo or inherited. if inherited then mum or dad is a carrier and future pregnancies would also carry high risk

Question 2

what should you be mindful of if a prenatal test used CVS as the sample?

Answer 2

CVS is from the placenta
There is always the possibility of confined placental mosaicism/post zygotic mitotic NDJ etc…

amniotic sample better, but ofc is risky, so is avoided e.g. by looking at the parents

Question 3

can you confidently exclude disorders/aneuploidies/mosaicism etc…?

Answer 3

No the thing is, excluding something just because you haven’t seen it is risky business - you can only do it ar a certain confidence level… the more cells you’ve looked at the more confident you can be

there’s a big table for this to tell you how many cells must be looked at in order to exclude a certain level of mosaicism at e.g. 95% confidence level

Question 4

How is the characteristic light and dark banding pattern in G-Banding generated?

Answer 4

G-Banding is achieved by applying the protease trypsin, which partially digests metaphase chromosomes, degrading histone proteins so that chromatin structures collapse. The chromosomes are then stained with Leishman’s dye (originally Giemsa, hence the “G” in G-Banding).

The resulting pattern (some areas light, some dark) reflects differences in chromatin structure, with euchromatin (less condensed) staining lighter and heterochromatin (more condensed) staining darker

Question 5

explain why euchromatin stains light and heterochromatin stains dark

Answer 5

trypsin’s digestion of histones causes chromatin collapse
Leishman’s dye is then applied, but collapsed chromatin is harder to access/the dye’s binding pocket is blocked
So more collapsed chromatin regions are stained less, i.e. appear lighter

euchromatin (GC rich) is transcriptionally active, and therefore has a more open conformation, while heterochromatin is more condensed

• since euchromatin regions start off as relatively open, they are more accessible to trypsin than more collapsed regions (heterochromatin).
• This means that following a partial digest, the open regions suffer greater collapse (more collapse, less access to dye, lighter staining)

and the opposite for heterochromatin

Question 6

why is the concentration of salt and length of exposure to trypsin so important for G-banding?

Answer 6

trypsin will digest all chromatin structures. It’s the idea that some regions (euchromatin) starting off as more open and therefore experiencing more collapse that results in the dye having different ‘strengths’ in colour

if you expose Chr to trypsin for too long, all the chromatin will experience more collapse, all less accessible to dye, you’ll get pale and fuzzy chromosomes, and possibly fall apart

Question 7

why is banding pattern highly reproducible?

Answer 7

euchromatin = GC rich, coding regions
heterochromatin = AT rich non-coding/LOW EXPRESSION

human genome highly conserved between individuals

Question 8

p arm vs q arm?

Answer 8

P arm = short
q arm = long (like a queue)

p arm is presented in a karyotype as above the centromere

Question 9

what phase should cells be in for karyotyping?

how do you encourage cells to be in this phase?

Answer 9

metaphase (condensed, and free in cytoplasm)

growth stimulation by hormones gets cell cycle growing, microtubule inhibitors prevent cell from entering anaphase. causing nuclei to swell by osmosis spaces out chromosomes

Question 10

why are cells fixed and what’s done next?

Answer 10

fixing kills the cells, freezing them in the cycle
also kills any possible pathogens in the sample

next, Chrs adhered to slide, exposed to sunlight at RT for 48 hrs, to denature proteins, remove residual fixative + enhance adherence, and to remove water to improve banding

Question 11

what are the three words used to describe a chromosome (based on centromere position)?

Answer 11

metacentric - centromere is roughly central (so P and Q arms are roughly same size

submetacentric - kind of classic image, with the p arms clearly shorter than the q arms (so centromere is higher than halfway)

acrocentric - typically small overall, the centromere is located near the end of the chromosome (also have satellited P arms, 13, 14, 15, 21, 22, Y)

Question 12

what’s special about acrocentric chromosomes?

Answer 12

all of their p arms are satellited, meaning they all contain repetitive DNA and rDNA genes - so loss of a p arm can often have no clinical consequences for the patient’s health, minus impacts on fertility

Question 13

what are the 7 groups of chromosomes?

Answer 13

Group A: Large metacentric chromosomes (1,2 ,3)

Group B: Large submetacentric chromosomes (4, 5)

Group C: Medium sized submetacentric chromosomes (6-12, and X)

Group D: Medium acrocentric chromosomes (13, 14, 15)

Group E: Short submetacentric (16, 17, 18)

Group F: Short and metacentric (19, 20)

Group G: Short and acrocentric (21, 22, Y)

Question 14

what is FISH used for/ useful for?

Answer 14

FISH uses fluorescently labeled oligonucleotide probes, that are complementary to
regions of genomic interest, to report on copy number (of large chunks that is) and positional information.

it is more sensitive than q-PCR

FISH also provides single cell/nuclear information, which is not possible for many
molecular genetic diagnostic technologies that require sample homogenisation.

It is
also possible to preserve nuclear morphology and tissue morphology during a FISH experiment, which means this technology is particularly useful for histological
preparations, useful in oncology

Question 15

explain how probes hybridise to sample DNA, explaining the concept of stringency in the process

Answer 15

nuclei harvested and adhered to slides like in G-banding…
in order for probes to be able to bind, DNA must be single stranded.

this depends on the stringency, which is like the energy available to break H-bonds sort of. it depends on heat and salt.
high heat = higher stringency.
low salt = high stringency - salt provides +ve ions that associate with the -ve DNA backbone, reducing the electrostatic repulsion between strands (so less salt = more repulsion = easier to separate)

so in FISH sample is denatured at 73 degrees (95 like in PCR is too high and would destroy morphology of chromosomes)

Question 16

what are the benefits of using cells in metaphase, or cells in interphase, for FISH?

Answer 16

metaphase -
- positional information (as you can see the morphology of a Chr a signal has colocalised on)

interphase -
- nuclei contain decondensed chromatin fibres and offered higher resolution
copy number information.
- Interphase FISH can also report if two genetic elements have
been fused together or separated by a rearrangement in the genome – important applications in diagnosis of cancers

in either case FISH is rapid, hence why it is used in prenatal diagnosis, or cases of leukaemia

Question 17

what kind of probes are there in FISH? (3/4)

Answer 17

Gene specific probes: Report copy number of disease critical regions e.g. Down
syndrome

• Centromeric probes: Complementary to alpha satellite sequences located in sub-centromeric regions of chromosomes. These probes tend to be very bright as the
  target regions are very large. copy number of 13, 18, 21
  X and Y in PND settings. very useful for detecting aneuploidy (^^Chrs named are common for aneuploidy)
• Telomeric probes and whole chromosome paints are very important to help geneticist to characterise chromosomes that have been observed to be abnormal by G-banding, e.g. inverted duplication

Question 18

give an example of gene-specific probes being used?

Answer 18

DiGeorge syndrome - often detected in infants after developmental delay, caused by a microdeletion, can cause morphological and heart abnormalities

Question 19

list some haematological malignancies in order of age prevalence

Answer 19

acute lymphoblastic leukaemia (ALL), Hodgkin Lymphoma, acute myeloid leukaemia, chronic myeloid leukaemia

Question 20

FR - Angelman Syndrome:
genetic cause/s?
symptoms?

Answer 20

Caused by the loss of the maternal UBE3A gene on chromosome 15
- this can be by deletion (70% of cases, deletion of approximately 4 million base pairs)
- or due to an imprinting issue… the paternal UBE3A gene is normally silenced due to imprinting, any paternal gene cannot compensate for the maternal one. Errors in imprinting or uniparental paternal disomy of Chr 15 can cause Angelman Syndrome.

Symptoms -
Developmental disabilities.
Seizures.
Speech deficits.
Motor dysfunction, including a jerky gait.
Characteristic happy and excitable demeanour, often with frequent laughter

Question 21

FR - Prader-Willi Syndrome:
- what is the genetic cause?
- what are the symptoms?

Answer 21

same region of chromosome 15, genes such as SNRPN encoding component of mRNA splicing, involve imprinting, this time the maternal allele is silenced.
deletion of this region on the paternal allele, or again uniparental disomy (maternal) could result in PWS

Symptoms -
Extreme feeding problems, including hyperphagia (insatiable appetite and obsession with food).
Developmental delay.
Decreased muscle tone leading to delayed motor development

Question 22

FR - Beckwith-Wiedemann Syndrome (BWS):

genetic cause?
Symptoms?
contribution to cancer?

Answer 22

genetic cause - uniparental disomy (two copies, for BWS when both come from the father), due to genes in area Chr 11p15 being imprinted.
Igf2 - normally only paternal copy is active
H19 - tumour suppressor, normally only mother’s copy is active.

two paternal Chr 15s mean double the Igf2 activity, so excess growth, and no H19 activity, loss of it’s tumour suppressor activity

symptoms - Overgrowth disorder with large birth size, Asymmetrical growth of body parts.
Increased risk of developing tumours, particularly Wilms’ tumour (kidney cancer)

risk in cancer -
Overexpression of Igf2 (insulin-like growth factor 2) drives excessive cell proliferation, increasing the risk of tumour formation.

Reduced expression of H19, a tumour suppressor gene, removes a critical control mechanism against cancer development.
Specific imprinting patterns in BWS patients are linked to particular tumour types

Question 23

FR - Emanuel Syndrome

what is the genetic cause?
what are the symptoms?

Answer 23

genetic cause - unbalanced translocation between Chr 11 and 22, has extra DNA (extra Chr 22, at least in part)

symptoms:
Symptoms include severe developmental delay,

intellectual disability,

weak muscle tone (hypotonia),

microcephaly,

craniofacial abnormalities,

heart defects,

ear anomalies,

failure to thrive

Question 24

if a dad was a carrier of a balanced t(11;22), why might his child have Emanuel Syndrome?

Answer 24

In a carrier of t(11;22), four chromosomal structures are present instead of (two copies of) two:

Normal chromosome 11
Normal chromosome 22
Derivative chromosome 11 (der(11))
Derivative chromosome 22 (der(22))
These four chromosomes form a quadrivalent structure instead of normal homologous pairs. This structure must properly segregate during meiosis to produce balanced gametes.

Step 2: 3:1 Meiotic Malsegregation
During anaphase I, the four chromosomes separate.
The most common error is a “3:1 segregation pattern”, where three chromosomes go to one gamete, and one goes to the other instead of an equal (2:2) split.
A common outcome of 3:1 segregation is that the sperm receives two normal chromosomes (one 11 and one 22) plus the derivative chromosome 22 (der(22)), while the egg contributes a normal 11 and 22

Question 25

how are PATRRs implicated in Emanuel Syndrome?

Answer 25

Palindromic AT-rich Repeat Regions (PATRR) are unstable DNA sequences, capable of forming secondary DNA structures such as cruciform, particularly in sperm cells during meiosis

they are found at specific breakpoint regions in multiple recurrent translocations, including 11q23 and 22q11. the secondary structures are susceptible to DNA breaks and when being repaired can result in a reciprocal translocation seen in unaffected carriers (who, as explained, are then likely to have a child with Emanuel Syndrome due to 3:1 mal-segregation)