Chromosome Abnormalities Flashcards
How is DNA packaged?
DNA wrapped around an octomer of histones
What does a octomer of histones consist of?
2 molecules each of H2A, H2B, H3 and H4
How many bp is in each octomer?
166
What is the purpose of histone H1?
It stabilises
How is higher order DNA structure established?
By hanging loops of DNA onto a protein scaffold to form chromatin
When are gene expressed?
When they form active chromatin
How can chromatin switch between active and inactive?
Epigenetic modification
How is chromatin converted from active to inactive?
By DNA methylation and histone deacetylation
How is chromatin converted form inactive to active?
DNA demethylation, histone acetylation
Where are changes made in activation and inactivation of chromatin?
To DNA and histones, not actual DNA sequence
What is being studies in chromosome analysis?
Metaphase chromosones
What does the study of metaphase chromosomes require?
Living cells for in vitro growth
What must be done to study metaphase chromosomes?
The cells must be cultured, accumulated at metaphase and harvested
Why does analysis of metaphase chromosomes take a long time?
Because it must be done stepwise
What are the steps in the analysis of metaphase chromosomes?
- Add spindle inhibitor
- Make hypotonic
- Add fixative
- Slides prepared and stained
- Visualised on light microscope
Give an example of a spindle inhibitor
Colcemid
What can be added to make the cell culture hypotonic?
Potassium chloride in varying concentrations
What fixative can be used in metaphase chromosome analysis?
3:1 methanol:acetic acid
What can the slides in metaphase chromosome analysis be stained with?
Giemsa stain
How are the slides from metaphase chromosome analysis interpreted?
Count, go through systematically to check all pairs, and check banding pattern is the same for each pair
What specimen types can be used for chromosome analysis?
- Bone marrow
- Blood (T-lymphocytes)
- Amniotic fluid
- CVS
- Solid tissue
How are bone marrow and blood for chromosome analysis grown?
In suspension
How long is bone marrow cultured for?
0-1 day
What long is blood cultured for?
2-3 days
Where is amniotic fluid, CVS and solid tissue grown for use in chromosome analysis?
On substrate
How long is amniotic fluid, CVS and solid tissue cultured for?
7-21 days
What do different sample types yield?
Chromsomes of differing lengths
How does the process of chromosome analysis differ depending on the sample type?
It doesn’t
What is true of chromosome analysis of all sample types?
Defined quality scores required to meet
How can chromosomes be analyses?
Karyotyping
What is karyotyping?
Systematic sorting of chromosomes
How is karyotyping carried out?
Metaphase chromosomes stained, paired up and grouped together
How are chromosome abnormalities described?
Using standard nomenclature ISCN
What are chromosomes composed of?
2 arms
What are the 2 arms of the chromosome called?
NAME?
What are the possible configurations of the chromosome arms?
- Metacentric- centromere in the centre, so p and q arms same length
- Submetacentric- centromere slightly more to one side, so p arm shorter than q arm
- Acrocentric- the centromere is the right at the end, so only the q arm is present
What does the p arm not contain in acrocentric chromosomes?
Euchromatic material, and therefore no genes of importance
What are chromosomes grouped based on?
Size and shape
What are the groups of chromosomes?
A → G
What groups are acocentric chromosomes?
D and G
What do groups D and G have on their p arms?
Satellites
Which of the chromosomes are not in size order?
22 is bigger than 21
What group is the X chromosome considered to be in?
C
What group is the Y chromosome considered to be in?
G
What methods of staining chromosomes are there?
- Solid staining
- G-banding
- C-banding
- Q-banding
- Replication
How is G-banding carried out?
NAME?
What is produced from G-banding?
Dark and light bands
Why are the metaphases exposed to trypsin?
It digests proteins differentially
Give 2 examples of Romanawksi type dyes?
NAME?
Which of the bands are gene rich in G banding?
The light G-ve bands
What are the dark G+ve bands in gene banding rich in?
AT
What are the light G-ve bands in gene banding rich in?
GC
What is shown in chromosome ideograms?
Standard banding pattern for each chromosome
How are the dark and light bands produced in chromosome ideograms numbered?
According to international convention ISCN
What does C-banding do?
Stains heterochromatin at centromeres, and 1, 9, 16 and Y q
When would the C-banding staining method be used?
If wanted to determine if change in chromosome is abnormality, or normal variations
What happens to normal chromosomes?
The chromosome lengths vary
What does Q-banding determine?
Y q variation
When would the replication staining method be used?
When testing for an active or inactive X in females
How can karyotyping be carried out faster?
Using automated or semi-automated systems
What happens in automated karyotyping?
The slides are scanned, images are taken, and digital karyotyping from images is carried out by cutting out the chromosomes and pairing them up on screen
What is the advantage of comparing chromosomes paired up on the screen?
Easier to compare chromosomes when lined up side by side
What is the problem with automated karyotyping?
It’s expensive and time consuming to create equipment
What is used to describe the karyotype in a chromosome report?
Standard ISCN format
How are chromosomes described using the standard ISCN format?
Chromosome number, sex complement and structural changes are given, separated by a comma
What is a chromosome report for a normal female?
46,XX
What is the chromosome report for a normal male?
46,XY
What is the chromosome report for a female with an extra chromosome 21?
47,XY,+21
What is the chromosome report for a male with a chromosome 7 inversion?
46,XY,inv(7)(p.11.2q11.3)
What happens in an inversion?
Segment between two break points is inverted
When are spaces found in the karyotype?
Never
What is the purpose of cytogenic analysis?
- Accurate diagnosis/prognosis of clinical problems
- Better clinical management
- Assess future reproductive risk
- Prenatal diagnosis
How can cytogenic analysis be involved in the accurate diagnosis/prognosis of clinical problems?
- Can identify the syndrome associated with an abnormality
- Accounts for the phenotype
- Accounts for pregnancy loss
Give an example of where cytogenic analysis can lead to better clinical management?
In the case of Klinefelter syndrome, where if detected before puberty, the person can lead a relatively normal life
What is Klinefelter syndrome?
When people carry 2 X chromosomes, as well as Y chromosome
When is the assessment of future of reproductive risk important?
When people already have an affected child
What future reproductive risk can be assessed using cytogenic analysis?
The risk of a live born abnormal child
What happens to the risk of having a Downs syndrome child after a previous Downs pregnancy?
It increases by 1%
Why is prenatal diagnosis important?
It provides information to clinicians and parents, who can then choose to terminate, or clinicians can plan for when the child is born
Why may a pregnant woman be referred?
- If the foetus displays constitutional abnormalities
- Infertility
- Recurrent fetal loss
What are constitutional abnormalities?
Abnormalities present from birth
Give 2 constitutional abnormalities
- Birth defects
- Abnormal sexual development
What acquired abnormalities may be reason for referral for cytogenic analysis?
- Leukaemia’s
- Solid tumours
What kind of leukaemia’s are there?
- Acute
- Chronic
- Myelodysplasia/myeloprolifierative disorders
What can specific translocation/abnormalities found in cancers give?
Prognostic information
How is Down syndrome screened for?
Maternal serum screening
How is maternal serum used to determine risk of Down syndrome?
Biochemical markers found in serum, as well as gestation and age are inserted into an algorithm, which can be used to determine chance
What happens if the chance of Down syndrome is found to be higher than 1 in 150 in a screening test?
An invasive test is offered
When is routine screening of foetuses performed?
First trimester
What happens in first trimester screening?
Test for biochemical markers and nuchal translucency on an ultrasound scan
What is happening as foetal scanning improves?
More abnormalities are found using ultrasound
Give 4 disorders that can be picked up on an abnormal ultrasound scan
- Cystic hygroma
- Cleft lip/plate
- Heart abnormality
- Limb abnormalities
What is a cystic hygroma?
A swelling at the back of the neck
What can be detected on prenatal DNA studies?
NAME?
What chromosome abnormalities can be detected prenatally?
FH chromosome abnormaility
How are DNA studies carried out prenatally?
DNA is directly extracted from chorionic villus
Give 5 examples of birth defects
- Dysmorphism
- Congenital malformations
- Mental retardation
- Developmental delay
- Specific syndromes
What can result from developmental delay?
NAME?
Give 3 specific syndromes that are birth defects
- Downs syndrome
- Williams syndrome
- DiGeorge syndrome
What is aneuploidy?
The loss or gain of whole chromosomes
What does aneuploidy result from?
Due to errors at cell division in meiosis
What are the two main types of aneuploidys?
- Trisomies
- Monosomies
Give 3 examples of trisomies
- Down syndrome
- Patau syndrome
- Edwards syndrome
What is the only viable monosomy?
Turners syndrome
What happens in Turners syndrome?
X-inactivation
What is polyploidy?
Gain of a whole haploid set of chromosomes
What is the most common of polyploidy?
Polyspermy
What is polyspermy?
The fertilisation of an egg by more than one sperm
How many pregnancies does triplody occur in?
2-3% of pregnancies
What % of miscarriages are caused by triploidy?
~15%
What happens to triploidy deliveries?
They die shortly after birt h
How many pregnancies does tetraploidy occur in?
1-2%
Where are tetraploid cells often found?
At prenatal diagnosis
What is the tetraploidy finding at prenatal diagnosis often caused by?
A cultural artefact caused by the failure of cell membranes to divide
What is seen in live births where babies die shortly after?
Diploid/triploid mosaicism
What does aneuploidy originate from?
Non-disjunction at one of the meiotic cell divisions
What does non-disjunction at one of the meiotic cell divisions form?
Gametes with a missing chromosome, and an extra chromosome
What affects the viability of gametes with missing or added chromosomes?
Which chromosome is involved
When can non-disjunction occur, other than in meiosis?
During mitotic cell division
What is the result of non-disjunction in mitotic cell division?
Mosaicism
What is mosaicism?
Two cell populations within an individual
What is meiosis?
The method by which we go to 46 diploid to 23 haploid
How can non-disjunction in meiosis occur?
NAME?
What normally happens when both chromosomes go into one cell in the first meiotic division?
Non-viable cell will be lost
What is the result when the cell is not lost when both chromosomes go into one cell in the first meiotic division?
2 gametes with 2 chromatids, and 2 with none
What is the result of both chromatids going into one cell at the second meiotic division?
Leaves one gamete with 2 chromatids, and one game with no chromatids
What can fertilisation of a gamete that has no or two chromatids with a normal gamete lead to?
Trisomy or monosomy
When may a pregnancy be viable despite the gametes being produced from meiosis where non-disjunction has occurred?
When the chromosome is 13, 18 or 21- the X must be involved
What happens in anaphase lag?
Chromosomes can be ‘left behind’ at cell division
What may cause anaphase lag?
Because of defects in spindle function or attachment to chromosomes
What can result from anaphase lag?
The lagging chromosome may be lost entirely in mitosis or meiosis
What is trisomy 21?
Down syndrome
What is the frequency of Downs syndrome?
1 in 650-1000
What is Down syndrome characterised by?
- Hypotonia
- Characteristic facial features
- Intellectual disability
- Heart defects
- Increased prevalence of leukaemia
- Increased prevalence of early Alzheimers
What is trisomy 18?
Edwards syndrome
What is the incidence of Edwards syndrome?
1 in 6000, with female predominance
What causes Edwards Syndrome?
Maternal meiosis 11 error
What is the medial lifespan of a Edwards Syndrome sufferer?
5-15 days
How are nearly all diagnoses of Edwards Syndrome made?
Prenatally
What are the symptoms of Edwards Syndrome?
- Small lower jaw
- Prominent occiput
- Low-set ears
- Rocker bottom ears
- Overlapping fingers
What is trisomy 13?
Patau syndrome
What is Patau syndrome?
Multiple congenital abnormalities, including polydactyl, and holoprosencephaly
What is the incidence of Patau Syndrome?
1 in 12,000
What is the chance of survival with Patau syndrome?
Majority die in neonatal period
What causes the majority of cases of Turner syndrome?
Absent paternal X
What do phenotypic differences in Turners syndrome depend on?
The parental origin of X
What are the symptoms of Turners syndrome?
- Short-stature
- Heart defects
- Mild learning defects
- Neck webbing
- Puffy feet
- Infertility
What is active in human cells?
Only one chromosome
What is the purpose of X inactivation?
Ensures that individuals have the same X chromosome complement that is active, as females have 2 X chromosomes, whereas males only have 1
What do X and Y chromosomes have in common?
Short regions at the tips of the long and short arms
What is at the short regions at the end of the P and Q arms of X and Y chromosomes?
Two pseudo-autosomal regions (PAR1 and PAR2)
What do PAR1 and PAR2 do?
Essential for pairing during mitosis
What will Turners syndrome patients be regarding the PARs?
They will be monosomic for the genes
What gene is found within PAR?
SHOX
What is the SHOX gene associated with?
Short stature
What is mosaicism?
Presence of 2 or more cell lines in an individual
What usually causes mosaicism?
Mitotic non-disjunction
Where is mosaicism found?
Either throughout the body, or tissue limited
What does the degree of mosaicism depend on?
When the error occured
What happens if the mitotic disjunction occurs in the first post zygotic division?
There is no mosaicism, and the disjunction looks like a meiotic event
How can mitotic non-disjunction lead to a non-mosaic karyotype?
In the first post-zygotic division, one cell gets 47,+N, and one gets 45,-N. The monosomy cell line is usually lost unless it involved X, as it is not viable, and the 47,+N then divides to produce 2 47,+21 gametes
What happens if the mitotic disjunction occurs in subsequent divisions?
There are 3 cell lines, of which the monosomy cell line is usually lost
The first 46,N cell divides into 2 46,N cells. If there is non-disjunction in one of those cell lines, the gametes are 27+N and 45,-N. The monosomy cell line is usually lost unless it involves the X chromosome
What is uniparental disomy (UPD)?
Presence of homologous chromosomes from one parent
What is isodisomy?
2 identical chromosomes from one parent
What is heterodisomy?
2 homolougous chromosomes from one parent
What is segmental UPD?
When only one part of the chromosome is involved
What is acquired UDP?
Solid tumours and leukemias
What do imprinted chromosomes show?
Differential expression of specific genes, depending on the parental origin of the chromosome
What is the effect of UDP is the chromosome involved is not imprinted?
It has no phenotypic efect
What chromosomes can UDP syndromes occur in?
6, 7, 11, 14, 15 and 16
Give 3 examples of UDP syndromes
- Prader-Willi (15)
- Russel-Silver (7)
- Beckwith-Wiedeman (11)
What are the 4 ‘common’ mechanisms to generate UDP?
- Trisomy rescue
- Monosomy rescue
- Gamete complementation
- Mitotic error
What do each UDP generation mechanisms require?
Two separate abnormal events
What is the most common mechanism of generating UDP?
Trisomy rescue
What happens in trisomy rescue?
If a disomic gamete from a meiotic error combines with a monosomic gamete, will get a trisomic conceptus. If then, during post-zygotic mitosis, a mitotic error occurs, there is a 1 in 3 chance of loosing the single chromosome from the monosomic gamete, leaving the 2 from the same gamete
What does trisomy rescue usually produce?
Heterodisomy
How can UDP be tested for?
Using molecular genetic testing, using information from repetitive DNA markers on imprinted genes/regions of interest
What does molecular genetic testing for UDP show?
Wether inheritance is bi-parental or not
What can be determined if a UDP test is confirmed?
It identifies the parental origin and allows the syndrome to be defined
What are the possible cytogenetic structural abnormalities?
- Translocations
- Inversions
- Deletions
- Duplications
- Insertions
- Rings
- Marker chromosomes
- Isochromosomes
What are the two types of translocations?
- Reciprocal
- Robertsonian
What are the two types of inversions?
- Paracentric
- Pericentric
What does the type of inversion that occurs depend on?
Wether the break point is within the same chromosome arm, or in both chromosome arms
What is generally true of marker chromosomes?
Generally quite small, and have a centromere that are not easily identifiable from banding pattern
What are isochromosomes?
Ones that are identical at both ends- mirror images
What are reciprocal translocations?
Two break rearrangements- there is a break in one chromosome, and a break in another, giving two segments of a chromosome that basically swap over
What are reciprocal translocation usually unique to?
A family
What is the exception to reciprocal translations being unique to families?
t(11;22)
What do carriers of reciprocal translations produce?
Balances and unbalanced gametes
What tends to happen if an individual carries a reciprocal translocation?
They are phenotypically normal
Why are reciprocal translocation carriers phenotypically normal?
All the chromosomal material is there, just in a different place
What is wether unbalanced offspring have abnormal phenotypes dependent?
Which regions are in trisomy, and which in monosomy
How can translocation imbalance be assessed?
Segregation analysis using a pachytene diagram
What are the 3 segregation types in reciprocal translocations?
- Alternate
- Adjacent 1- non homologous centromeres
- Adjacent 2- homologous centromere
What is the most common form of segregation that gives imbalance?
Adjacent 1- non homologous centromeres
What can be produced from adjacent 2?
3:1 disjunction and 4:0 disjunction, both of which are unbalanced
What does translocation form?
Quadrivalent
What is produced when segregation is alternate?
Either two normal chromosomes, or two derivative chromosomes
What needs to be done to assess unbalanced segregant outcomes?
- Establish likely segregation
- Check to see if imbalances have been reported before
- Quote risks if established
What causes Robertsonian translocations?
Two acrocentric chromosomes fusing together
Which are the acrocentric chromosomes?
13, 14, 15, 21 and 22
What kind of Robertsonian translocations can be produced?
Mono or dicentric
What is the most common Robertsonian translocation?
13;14
What is the chromosome count in balanced carriers of Robertsonian translocations?
45
Why is the chromosome count 45 in balanced carriers of Robertsonian translocations?
Because two chromosomes are fused together
What is formed at meiosis in Robertsonian translocations?
Trivalent
Are trivalents stable?
No
What is the risk with Robertsonian translocations?
Aneuploidy risk
Who is at higher risk from Robertsonian translocations?
Females
What are homologous carriers of Robertsonian translocations unable to do?
Have normal pregnancies
Why can homologous carriers of Robertsonian translocations have normal pregnancies?
Because they can only pass over the Robertsonian chromosome
What do deletions arise through?
Uneven pairing and recombination during meiosis
What are the two types of deletions in chromosomes?
NAME?
Where does a terminal deletion affect?
The ends of the chromosome
Where does an interstitial deletion affect?
Within the arms of the chromosome
What is required to diagnose or confirm a microdeletion?
FISH technique
Why is the FISH technique required to diagnose or confirm microdeletions?
Because they can’t always be seen on G bandings, because the resolution of a G banded karyotype is around 5-10M (depending on what sample type you’re looking at), and some microdeletions are only around 100Kb in size
What must be done when reporting abnormal results of chromosome analyses?
- Give correct ISCN
- Describe abnormality in words and what it means
- Balanced or unbalanced
- Is there monosomy/trisomy for a region
- Relate to clinical problems
- Request parental/family samples if required
- Refer to clinical genetics
- Provide appropriate literature if available
What kind of technique is FISH?
Molecular cytogenetic
What does FISH allow?
The answering of specific questions
What is a requirement for FISH?
Need to know what we’re looking for
What is used in FISH?
Probes for specific chromosome or loci
How is FISH carried out?
- A DNA probe is flourescently labelled
- Target material is added to DNA probes, and heat to denature probe and chromosomes to single strand
- Hybridisation by reannealing at 37º c .
- Washing to remove unbound probe
- Visualisation under fluorescence microscope
What are the 4 types of probes used in FISH?
- Locus/gene specific probes
- Centromere probes
- Telomere probes
- Whole chromosome paints
What are locus/gene specific probes used for?
Detecting microdeletion/duplications syndromes that are too small to see on G-banded chromosomes
What size are centromere probes?
Large
What is the advantage of centromere probes being large?
Easy to see
What are centromere probes used for?
- To identify the chromosome of origin
- Visualise metaphase and interphase
- Copy number analysis
- Identifying derivative chromosomes and markers
What are whole chromosome paints used for?
To identify a chromosome in rearrangement
What is prenatal aneuploidy screening (PND) analysing?
Interphase
What can be used for PND?
Cultured cells or uncultured cells
How long are cells cultured in PND?
Up to 14 days
What does PND culturing cause?
Anxiety
What is used in uncultured cells in prenatal aneuploidy screening?
FISH probes for 13, 18, 21, X and Y
What areFISH probes for 13, 18, 21, X and Y used in prenatal aneuploidy screening?
Because these are common aneuploidies
How quickly can a result for prenatal aneuploidy screening be obtained?
- For those testing using FISH probes, 24-48 hours
- A full karyotype in 14 days
What is the concordance rate of prenatal aneuploidy screening using the FISH probes compared with the full karyotype?
99+%
What does microarray examine?
The whole genome at high resolution
What can be looked for using microarray?
Copy number changes
What can’t be detected using microarray?
Balanced rearrangements or mutation
What does microarray compare?
Patient DNA to normal control DNA
What are the advantages of microarray technology?
High resolution
Lots of different genetic conditions can be looked at at once
Automated
Get detailed information about the genes
What are the disadvantages of microarray?
Cost compared to karyotype
Will not detect balanced rearrangements , copy number variation and what is abnormal
Mosaicism may be missed
What can cause problems with microarray technology?
Variation of uncertain significance
Why does variation of uncertain significance create a problem?
Because there could be a novel pathogenic call (not seen before), a novel call with no genes in that region or a novel call that doesn’t fit with the patient phenotype
What does non-invasive prenatal resting use?
Cell free foetal DNA found in the maternal plasma from 9 weeks gestation
What is the problem with non-invasive prenatal testing?
Technically challenging
DNA quality deteriorates quickly
How is the problem of the quality of DNA deteriorating quickly in prenatal genetic testing overcome?
Using special tubes to stabilise sample
What techniques are used for non-invasive prenatal testing?
Digital PCR
Next generation sequencing
What does NIPT reduce?
The need for invasive testing
What may invasive testing be needed?
Confirmation of abnormal NIPT
What aspect of NIPT has been approved?
NIPT testing for single gene disorders
What does next generation sequencing allow?
Fragmenting of genomic DNA
What is the advantage of fragmenting of genomic DNA?
So it can all be sequenced at the same time
What does next generation sequencing create?
A lot of data which needs to be protected and stored carefully
What is next generation sequencing suitable for?
Multiplexing with biological barcodes
What is used to conform NGS findings?
Sanger sequencing
What is the advantage of NGS sequencing?
It reduces the cost of testing individual genes by offering panels of gene testing
What is whole exome sequencing?
The targeted sequencing of al exons
What must be done in whole exome sequencing?
Identify de novo mutations in trio analysis
