1
Q
What is cytogenetics?
A
Study of chromosomes (no., structure, instability etc)
2
Q
List 3 types of chr abnormality
A
- Chr rearrangements
- Whole chr aneuploidy
- Copy no. imbalance
3
Q
What are chr rearrangements?
A
Bits of chr move to diff chr so have right amount of genetic material but in wrong place
4
Q
What is whole chr aneuploidy and give e.g.
A
- whole chr is add/missing
e. g. Trisomy 21/Down’s Syndrome
5
Q
What is copy no. imbalance?
A
Bits missing from/extra bits within chr
6
Q
List techniques used in traditional cytogen and what they involve
A
- G-banding (Chr banded to give specific pattern)
- Some FISH
- Breakage
Cell culture required to collect metaphase cells, then look at through microscope
7
Q
List techniques used in molecular cytogen and what they involve
A
- QPCR (quantitative flourescent)
- MLPA
- Array CGH
Extract DNA from patient then tests carried out on DNA
8
Q
What can chr rearrangements lead to?
A
- recurrent miscarriage
- infertility
9
Q
What can copy no. imbalance lead to?
A
- dysmorphism
- dev delay
- learning diff
- specific phenotypes e.g. epilepsy, diabetes, cardiac malformations
10
Q
What can chr breakage syndromes lead to?
A
- fanconi anaemia
- ataxia telangiectasia
11
Q
How does whole chr aneuploidy arise?
A
- following non-disjunction at mitosis/meiosis
12
Q
What does whole chr aneuploidy lead to?
A
large genomic imbalance leads to loss of conceptions except where chr involved is gene poor ( trisomy 13, 18 + 21 are only autosomal trisomies that are found at live birth)
13
Q
Briefly describe what happens to chr in meiosis I
A
- Replication - homologous pairs replicate to form sister chromatids
- Synapsis - sister chromatids synapse (come next to each other)
- Recombination - crossing over
- Disjunction - split into 2 daughter cells
14
Q
Describe what happens if there’s non-disjunction in meiosis I
A
- instead of sister chromatids splitting into 2 daughter cells, go into same daugther cells
- in meiosis II, end up with 2 disomic daughter cells with an extra chr and 2 nullisomic daughter cells with no chr
15
Q
List 3 conceptuses that can result from non-disjunction
A
- disomic gamete + normal gamete –> trisomic conceptus
- nullisomic gamete + normal gamete –> monosomic conceptus
- disomic gamete + nullisomic gamete –> uniparental disomy (both chr from same parent)
16
Q
What is mosaicism?
A
Diff cells in same indiv have diff no.s/arrangements of chr dev from single fertilised egg e.g. indiv with mosaic trisomy 18 has some cells with 3 copies of chr 18 + other cells with 2
17
Q
How does mosaicism arise?
A
in an initially normal conceptus, due to non-disjunction/anaphase lag
18
Q
What is anaphase lag?
A
- delayed movement of 1 homologous chr/chromatid + fails to connect to spindle/not drawn to pole properly
- so not included in reforming nucleus and lost from cell
- chr not in nucleus of 1 of daughter cells so get 1 normal daughter cells and 1 with monosomy
19
Q
List 3 types of mosaicism
A
- Somatic
- Gonadal
- CPM
20
Q
What is somatic mosaicism?
A
occurs when somatic cells of body are of more than 1 genotype arising from single fertilised egg due to mitotic errors at 1st/later cleavages
21
Q
What is somatic mosaicism likely to result in?
A
abnormal phenotype if there’s high prop of abnormal cells
22
Q
When is somatic mosaicism not as severe?
A
if phenotype improved by normal cell type i.e. mosaic trisomy 21 not as severe as full trisomy 21
23
Q
What is gonadal mosaicism?
A
Some gametes carry mutuation but rest are normal
24
Q
How does gonadal mosaicism arise?
A
during formation of germ cells + usually only identified following 2 pregnancies with same de-novo abnormality
25
What does gonadal mosaicism lead to?
abnormal cell line in gonads but normal somatic cells + so manifests as repeated conceptions with same aneuploidy
only some children affected even for dom disease
26
What is CPM?
Confined Placenta Mosaicism
| - confined to embryonic tissue and occurs in 1-2% placenta and so may go undetected
27
What can CPM result in?
- may have normal outcome/compromised function of placenta or abnormal cell may go to placenta and so not in foetus
- may result in UPD following trisomy rescue
28
List 4 types of chr rearrangements
1. Robertsonian translocations
2. Reciprocal translocations
3. Inversions
4. Intrachromosomal insertions
29
How do Robertsonian translocations arise?
Fusion of 2 acrocentric chr (centromere near top)
30
What is structure of acrocentric chr?
- centromere near top
- short arms have little satellite DNA with no important gen function in terms of chr imbalance so can miss entire short arm + no phenotypic consequence
31
What is prevalence and most common fusions of Robertsonian translocations?
1 in 1000
| der (13;14) + der (14;21)
32
What are phenotypes of balanced carriers of Robertsonian translocations?
- phenotypically normal (as short arm insig) but have rep risks:
- recurrent miscarriages
- Patau and Down's syndrome
- male infertility (as it interferes with spermatogen)
33
What are the 2 types of Robertsonian translocations seg?
1. Alternate Segregation
| 2. Adjacent Segregation
34
What happens during alternate seg of Robertsonian translocations?
During meiosis, Robertsonian chr (e.g. fusion of chr 14 + 21) goes into 1 daughter cell
sep chr 14 + chr 21 go into other daughter cell
but both cells have balanced genome
35
What happens during adjacent seg of Robertsonian trnaslocations?
Rob chr and chr 21 go into 1 daughter cell which gives rise to trisomy 21
Chr 14 goes into another daugther cell
For F carriers of 14;21 Robertsonian, 15% risk of trisomy 21 to offspring
36
What are reciprocal translocations?
Exchange of material between any segments of 2 non-homologous chr
37
What is the prevalence and 1 main feature of reciprocal translocations?
1 in 500
| almost always unique to 1 family
38
Which reciprocal translocation is recurrent and why?
46, XX, t(11;22) - maybe bc there's higher prob of it occuring de novo/founder effect
39
What are the phenotypes of balanced carriers and risks of reciprocal translocations?
phenotypically normal but rep risks dependant on size of translocated segments:
- infertility
- miscarriage
- child with congenital abnormalities
40
List 3 types of alt seg of reciprocal translocations
- Normal and Balanced: 2 normal chr go into one daughter cell and 2 reciprocal chr go into other daughter cell
- Both unbalanced: normal + abnormal chr go into both daugther cell (get too much of 1 chr in each)
- Both unbalanced: 1 normal chr in 1 daughter cell + 2 abnormal + 1 normal chr go into other daughter cell
41
List 2 types of chr inversions
1. Pericentric
| 2. Paracentric
42
What is pericentric inversion?
- Crossing over in inverted region causes 2 copies of short arm + none of long arm and vice versa
- includes centromere
43
What is paracentric inversion?
- Inverted segment doesn't include centromere
| - chr form inversion loop + crossover in loop
44
Why are paracentric inversions benign during meiosis?
Don't give rise to viable imbalances so pregnancy not compromised
45
What is FISH?
- Flourescent In Situ Hybridisation
| - diff types of probe with range of applications
46
Describe how FISH works
1. Have target DNA + DNA probe complementary to target sequence
2. Denature target DNA (sep double strand) + DNA probe anneal to target sequence which is now tagged with fluorescent marker
47
What other techniques can be used for FISH?
Sub-telomere probes for long + short arm
Whole chr paints
M-banding = use colour to see rearrangements
e.g. use control probe locus + TUPLE locus 1 present on chr, can confirm diagnosis
48
What is pre-natal cytogenetics?
Traditional tests using G-banded chr
49
What is the disadv of using pre-natal cytogenetics?
approx 2 weeks for results (amniocentesis)
| results needed more quickly for reassurance/pregnancy management so use FISH, QF-PCR
50
How would you count chr using QF-PCR?
no. of peaks = ratio
height of peak = length of marker
e.g. disomy 1:1 (2 normal chr) prod 2 equal peaks, trisomy 2:1 (1 peak twice as high as other), trisomy 1:1:1 (3 equal peaks) and uniformative 1:1/1:1:1 (single peak)
51
Give e.g.s of segmental copy no. imbalance
normal copy no. = 2 (except for sex chr) so 2 copies of genome for each chr
- deletions (1 copy of gene on 1 chr/none on both)
- duplications (1 copy on 1, 2 on another so 3 in total)
- triplications (4 in total)
52
Why can't G-banded chr analysis be used to find chr imabalances?
Has res of approx 5-10 Mb and many imbalances (mirodel) cause syndromic disease so can't be picked by G-banded
53
Describe how array cGH works
1. Chop up DNA and label with fluorescent marker so they're all labelled
2. Do the same to ref DNA and mix them together and apply to microarray slide
3. DNA probes bind to complementary DNA sequences
54
What is an array?
many DNA probes attached to glass slide
55
How can arrays be read?
- Genomic balance means equal mix of green and red
- e.g. Red spot means more test material compared to control so duplication in patient's genome
- e.g. green spot means more control material compared to test so deletion in patient's genome
56
What can array cGH detect?
- whole chr aneuploidy
- microdel/dup syndromes
- subtelomere imbalance
- other regions of imbalance (copy no. variants) but unsure if benign/pathogenic
57
How can you ascertain clinical consequence of of prev unreported imbalance?
- inheritance
- no. of genes (burden)
- specific gene content
58
How can you ascertain clinical consequence of of prev unreported imbalance using inheritance?
- de-novo more likely to be pathogenic
- inherited from affected parent - more likely to be pathogenic
- inherited from unaffected parent - more likely to be benign
59
How can you ascertain clinical consequence of of prev unreported imbalance using no.of genes?
greater no. of genes means greater burden and more likely to be cause
60
How can you ascertain clinical consequence of of prev unreported imbalance using specific gene content?
whether specific genes can be correlated to phenotype
61
What have research studies using array cGH shown?
normal indiv carry multiple CNVs
62
What are copy no. variants?
sections of genome that's been repeated and no. varies between indiv
63
What may diff combos of CNVs contribute to?
Phenotypic variation between indiv
64
Why doesn't a "normal" phenotype really exist?
Every locus has imbalance within normal pop
65
What can be said about many patients genes?
Many patients have imbalance for genes of unknown function/genes whose function is unrelated to referral indication
Some of genes may be ass with other clinical phenotypes/may predispose to malignancies
66
Give an e.g. of the effects of CNVs on protein complexes
- if have protein complex with red and green subunits
- CNV 1 can remove green subunit but still have normal phenotype as protein can still function provided its got at least 2 components
- if CNV2 removes red subunit - causes phenotype as protein now can't function
67
What is a feature of smaller size CNVs?
Have greater % of inheritance
68
How has genetic testing changed over time?
FISH, G-banding, MPLA replaced by array CGH but doesn't give info about structure so replaced in future by whole exome/genome sequencing
69
What are microdeletion syndromes?
Submicroscopic chr deletions 100kb-3000kb
| Are syndromes bc they occur at low freq in all pop
70
What causes microdel syndromes?
- Genomic structure at disease locus - predisposes to gene deletion - duplication by unequal recomb
- Mediated by low copy repeats - where unequal crossing over within LCR
- LCR don't line up underneath each other as normal
- rep goes through 1 block then crosses over to other strand which leads to dup
71
List e.g.s of microdel syndromes
- Di George - del (22q)
- Williams - del (7q)
- Angelman - del (15q)
- Prader-Willi - del (15q)
- Wolf-Hirschhorn - del (4p)
