2. Chromosomes (2) Flashcards
What is a karyotype? (5)
▣ A karyotype is an individual’s collection of
chromosomes
▣ It is the number and appearance of chromosomes in
the nucleus of a eukaryotic cell
▣ Look at length, position of centromeres, and
banding patterns
▣ Look for differences from “normal”
▣ It involves the isolation, staining and visual
examination of chromosomes
What are the advantages of a Karyotype? (4)
- Detect large numerical and structural abnormalities
- Able to visualise all of the chromosomes in a single test
- Can detect balanced and unbalanced chromosome rearrangements
- Can detect mosaicism
What are the disadvantages of a Karyotype? (7)
- Requires fresh tissue
- Time consuming
- Must be done on cultured cells
- Can’t detect small deletions/duplications/
rearrangements - Can’t identify marker chromosome origins
- Can’t identify single gene defects
- Accuracy depends on experience of the
cytogeneticist
QF-PCR
▣ Quantitative Fluorescence-Polymerase Chain Reaction
▣ Detection of common aneuploidies: (5)
□ Trisomy 13
□ Trisomy 18
□ Trisomy 21 (Down syndrome)
□ Turner syndrome (45, X)
□ Klinefelter syndrome (47, XXY
QF-PCR
▣ Uses markers on chromosomes: (5)
13, 18, 21, X and Y
QF-PCR
▣ Utilises _______ that target specific short tandem repeats (STRs) on Chromosomes 13, 18, 21, X and Y.
▣ PCR _______ generates products that differ in length depending on the number of repeats
▣ TAT ± 2-3 days
primers
amplification
QF-PCR advantages: (3)
- Quick turnaround time
- Cheap test
- Does not require cells to be cultured
QF-PCR disadvantages: (3)
- Limited to only detect common aneuploidies
- Not designed to detect structural abnormalities
- Mosaicism may be missed
What is FISH? (6)
▣ Fluorescent in-situ hybridisation
▣ Detects small deletions/duplications which are not visible by karyotype
▣ Molecular cytogenetic method used to detect and localize the presence or absence of specific regions on
chromosomes
▣ Fluorescent probes hybridise to specific regions on the chromosome
▣ Allows us to detect micro-deletions and duplications but you need to know what you are looking for to know which
probes to use.
▣ Can detect rearrangements of chromosome
What are the advantages of FISH? (3)
- Greater resolution than Karyotype- can detect microdeletions and duplications
- Can detect structural rearrangements
- Can detect balanced rearrangement
What are the disadvantages of FISH? (3)
- Targeted approach- need to know what you are looking for
- Need to design probe specific to region of interest
- Usually reserved for detection of common microdeletion and duplication syndromes
What is MLPA? (6)
MLPA advantages: (2)
- Better resolution than karyotype - can detect microdeletion and duplication syndromes
- Does not require cells to be cultured
MLPA disadvantages: (3)
- Will miss regions of chromosome not covered by probes
- Cannot detect balanced chromosome rearrangements
- Cannot visualise chromosomes and can therefore not always elucidate mechanism of rearrangement
Which method has the highest resolution?
Microarray
▣ 2 different types:
□ Comparative genomic hybridization (CGH) array
□ Single nucleotide polymorphism (SNP) arra
What is a microarray? (3)
▣ High resolution test
▣ Assesses chromosomal gains and losses
▣ Looks at all chromosomes at once
What is the Comparative Genomic Hybridization
(CGH) Array? (2)
What is the Single Nucleotide Polymorphism
(SNP) Array? (2)
What is the function of CGH-Arrays? (5)
▣ Method for genome-wide detection of DNA copy-number variants (CNV)
▣ Compares the patient’s genome against a reference genome
▣ Identifies regions of genomic imbalance
▣ Resolution: 10 000 bp
▣ 10-15% increased diagnostic yield over traditional cytogenetic methods
How are CHG arrays sampled? (5)
▣ Patient sample and the reference sample - labelled
with different fluorescent dyes
▣ The two samples are applied on an array, and
complementary sequences bind. Colour/Intensity
information is collected by a scanner.
▣ Where there is no change in the sequence copy
number in the test (patient) sample, there will be
equal binding of test and reference sample DNA, and
equal amounts of each coloured fluorescence will
produce a net emission colour (yellow).
▣ For sequences where there has been a duplication in
the test sample, there will be more red than green
fluorescence and an overall red emission
▣ Deletions will result in a reduced level of red
fluorescence relative to the green fluorescence from
the reference sample, and a net emission of green
light
What are the advantages of CGH arrays? (6)
- Does not need to be done on cultured cells
- Requires very little genomic DNA
- Analyse all chromosomes in one experiment
- No targeting needed
- Detects numerical changes and unbalanced structural changes
- Increased resolution and diagnostic yield
What are the Disadvantages of CGH arrays? (5)
- Does not detect balanced rearrangements
- Cannot visualise the chromosomes and therefore elucidating mechanism of rearrangement may be difficult
- Deletions and duplications within regions of minimal probe coverage will be missed
- Low-level mosaicism wont be detected
- Polyploidy cannot be detected
CGH-arrays
Test Indications
Microarray should be first tier testing in patients with: (7)
▣ Unexplained learning difficulties
▣ Intellectual disabilities/cognitive impairment
▣ Developmental delay
▣ Behavioural problems including autism spectrum disorders
▣ Dysmorphism
▣ Multiple congenital abnormalities
▣ Certain isolated congenital abnormalities (cardiac defects, diaphragmatic hernias)
Challenges with newer technologies: (5)
▣ Incidental findings
□ Do we report them or not?
▣ Variants of uncertain significance (VUS)
▣ Pathogenic versus normal genetic variation?
▣ Reference sequences are constructed mainly from Caucasian populations
▣ African populations are under researched
How do you know when to use different methods?
QF-PCR
Karyotype
FISH
MLPA
Microarray
Choosing the correct test
1. Six-month-old baby presenting with clinical features suggestive of trisomy 21
a. QF-PCR
b. Karyotype
c. FISH
d. MLPA
e. Microarray
a.
Choosing the correct test
2. Four-year-old who presents with developmental delay, a cardiac defect, and
dysmorphic features
a. QF-PCR
b. Karyotype
c. FISH
d. MLPA
e. Microarray
e.
Choosing the correct test
3. A suspected unbalanced translocation was detected on microarray in a baby with multiple congenital abnormalities. The CNVs detected included a 15Mb
terminal deletion on chromosome 10 and a 38Mb terminal duplication on chromosome
- What test would you do on the parents to see if one of them may carry a balanced translocation?
a. QF-PCR
b. Karyotype
c. FISH
d. MLPA
e. Microarray
b.
Choosing the correct test
4. A suspected unbalanced translocation was detected on microarray in a baby with multiple congenital abnormalities. The CNVs detected included a 1.5Mb
terminal deletion on chromosome 10 and a 3.8Mb terminal duplication on chromosome
- What test would you do on the parents to see if one of them may carry a balanced translocation?
a. QF-PCR
b. Karyotype
c. FISH
d. MLPA
e. Microarray
c.
Choosing the correct test
5. Six-year-old with a suspected diagnosis of 22q11.2 deletion syndrome, a common microdeletion syndrome
a. QF-PCR
b. Karyotype
c. FISH
d. MLPA
e. Microarray
c,d,e
What is Prenatal testing? (2)
- Testing for diseases or conditions in a fetus before it is born
- Main goal:
- Perform prenatal diagnostic testing at the earliest possible gestation
Types of prenatal tests: (2)
- Chorionic villus sampling
* 11 – 14 weeks gestation
* Cells from chorionic villi of the placenta - Amniocentesis
* 18 – 23 weeks
* Fetal cells in amniotic fluid
What is Aneuploidy? (2)
- Loss or gain of one chromosome from a homologous pair
- Caused by non-disjunction
What is monosomy vs trisomy? (2)
- Karyotype
* Cell culture and _________
* CHROMOSOME staining and banding
* TAT ± 2-3 weeks
* _______ and time-consuming
* Gold standard
harvesting
expensive
- QF-PCR
* Use ________ dye-labelled primers
* Target specific SHORT TANDEM REPEATS
* Amplify the STRs via ____
* Capillary ___________
* TAT ± 2-3 DAYS
Fluorescent
PCR
electrophoresis
- Short tandem repeats
* Also called ____________
* Tandemly repeated nucleotide units of 2 – 6
base pairs (bp)
* STR loci account for ± 3% of the human
genome
* Most STRs are found in ____-______ regions of
genes (± 8% are located in coding regions)
* In humans, chromosome ____ has the highest
density of STRs
* The most common STRs in humans are ___-_____.
microsatellites
non-coding
19
A-rich
What are STRs designed to do? (3)
- Primers are designed to flank the STRs
- Primers for different STR loci are labelled with different
coloured dyes - PCR amplification generates products that differ in
length depending on the number of repeats
Naming of STRs
Example: D3S1266
D
3
S
1266
D represents DNA
3 means chromosome 3
S stands for STR
1266 is the unique identifier.
Aneuploidy kits:
The process:
1. Collect sample – __________/CVS/blood
2. Extract DNA
3. Amplify DNA – QF-PCR
4. Perform _______ electrophoresis
* Using a genetic analyser
5. _____________
Analyse and interpret data
amniocentesis
capillary
Electropherogram
What is the result of the fetal aneuploidy – sex and diagnosis?
