Microarrays in genomics Flashcards
(35 cards)
1
Q
What is a constitutional genetic disorder? (2)
A
- Genetic aberrations which are present in all cells from birth (also called germline)
- Somatic genetic aberrations are acquired later in life and are mosaic
2
Q
What is the resolution of karyotyping?
A
5-20 megabase
3
Q
What is the principle of karyotyping? (3)
A
- Trypsin digestion produces light and dark bands
- Dark = AT rich, heterochromatin
- Light = GC rich, euchromatin
4
Q
How does FISH work? (6)
A
- ssDNA probe complimentary to target sequence is fluorescently labelled
- Denature the chromosomes to allow the probe to access the target sequence
- Hybridise
- Wash to remove background signal
- Apply DAPI to visualise chromosomes
- Analyse with fluorescent microscope
5
Q
What are the types of FISH probe? (2)
A
- Whole chromosome paint
- Locus specific probe
6
Q
What can FISH show? (3)
A
- Gene amplification
- Translocations
- Gene fusion
7
Q
What is a microarray? (5)
A
- A set of DNA probes are attached on an array (slide)
- Assay uses fluorescently labelled patient DNA to bind to the probes
- Compare with relative quantity of control DNA to identify gain and loss of chromosomal material
- Gains and losses = copy number variants
- Use software to identify the genomic location of the CNVs and which genes are involved
8
Q
What is the benefit of karyotyping over microarrays?
A
Microarrays can’t detect genetically balanced rearrangements e.g. translocations
9
Q
Which patients are tested using microarrays? (3)
A
- Children with developmental delay (autism, intellectual disability, dysmorphic features, congenital abnormalities)
- Prenatal analysis (DNA from amniotic fluid/CVS, abnormal ultrasound scans)
- Cancer (e.g. myeloid dysplastic syndrome loss of 5q)
10
Q
What are the benefits of microarrays over karyotyping? (3)
A
- Microarrays have increased sensitivity: karyotyping can only detect gains and losses of 5-10 megabases, microarrays can detect gains and losses of 100 kilobases
- Karyotype of children with developmental delay has a diagnosis rate of 5% but testing the same children with microarrays has diagnosis rate of 20%
- Higher sensitivity = more diagnosis
11
Q
What are the 2 main types of microarrays?
A
- Oligonucleotide arrays
- Single nucleotide polymorphism (SNP) arrays
12
Q
What are oligonucleotide arrays?
A
Uses oligonucleotide DNA probes that are complimentary to specific regions of the genome
13
Q
What are SNP arrays?
A
Uses 100 000s of SNP probes across the genome to measure the ratio of one allele vs another by targeting the normal variant within the human genome
14
Q
How do oligonucleotide arrays work? (7)
A
- DNA is fragmented
- Patient and control DNA have different fluorescent labels
- Competitive hybridisation to the oligonucleotides on the slide
- The amount of bound patient vs control DNA is measured by relative fluorescence intensity
- If patient and control is the same amount, relative fluorescence will be 1:1
- If patient has stronger fluorescence (1:2) this means the patient has a gain of genetic material at the location the oligo probe is specific for
- If the patient has weaker fluorescence (2:1), the patient has lost genetic material at this location
15
Q
What do oligonucleotide arrays detect? (2)
A
- Copy number variants (CNVs)
- Do not detect genetically balanced rearrangements e.g. translocations and inversions
16
Q
When are apparent losses/gains significant on an oligonucleotide array profile? (2)
A
- At least 3 neighbouring probes showing a loss or gain
- Single calls are likely to be artefacts
17
Q
What is an SNP? (6)
A
- Single nucleotide polymorphism
- A change in a single nucleotide in the DNA sequence of the human genome
- Not mutations, normal variants
- Approximately 10 million SNPs in the genome
- In a given location where there is a SNP there are 2 allele types: one with SNP and one WT referred to as A and B
- Individual can be WT for both alleles or have SNP in both or heterozygous (AA, BB, AB, BA)
18
Q
How do SNP arrays work? (9)
A
- Only patient DNA required, no control
- Contains DNA probes for WT and known SNPs
- Each allele (A and B) are represented on the array
- SNP A and B probes are differently fluorescently labelled red or green
- Amplify and fragment patient DNA, hybridise
- Patient DNA binds the probes = fluorescence emitted and detected by scanner
- Both probes emit fluorescence = heterozygous, one = homozygous
- Can also identify gains/losses
- Patient may be AA, BB or AB
19
Q
What is loss of heterozygosity? (4)
A
- Can be detected by SNP array
- Loss of small or large chromosomal region resulting in no heterozygous SNPs
- Can imply that the individuals’ parents are related as it shows there is less normal variation in their genotype
- LoH can unmask recessive mutations that may cause disease
20
Q
How do you interpret the data from SNP array? (5)
A
- Middle band = AB heterozygous, 1:1 fluorescence
- Top band = AA homozygous, 1
- Bottom band = BB homozygous, 0
- SNPs are plotted in order along each chromosome
- Gap in the middle = centromere
21
Q
What does a deletion look like on a SNP array? (3)
A
- Deletion means only one allele so there can only be an A allele or a B allele, can’t be a AB heterozygous genotype
- Large run of homozygosity indicates a deletion
- Would need to see many areas of homozygosity to consider LoH
22
Q
What does a duplication look like on a SNP array? (3)
A
- 2 bands in the middle rather than 1 because each SNP will either be AAA, BBB, AAB or ABB (depending which is duplicated) but no AB
- 4 regions of calls with different relative fluorescence
- Seen in trisomy
23
Q
What is Pallister-Killian syndrome? (7)
A
- Characterised by mosaic tetrasomy isochromosome 12p
- Never seen in blood karyotype, can be seen in skin cell karyotype
- Non-mosaic isochromosome 12p is lethal
- Developmental delay and intellectual disability
- Extra digits
- Characteristic facial features
- Hypotonia
24
Q
Is a gain or loss more likely to have a phenotypic consequence?
A
Loss because the absence of a protein is more significant than having too much of a protein (deletion is a more common mechanism of disease)
25
How should a CNV be assessed? (6)
- Size isn't a reliable indicator of predicting normal gain/loss or pathogenic gain/loss because normal CNVs can be very small or very large
- Consider whether any genes are involved: may not contain any genes (unlikely to be pathogenic), may contain a gene associated with a genetic disorder
- Consider location: the gain/loss location may be associated with a particular genomic syndrome
- Do the genes affected by the CNV fit with the referral reason
- Is the CNV present in the normal population (if the CNV is in more than 1% of the population we can dismiss it as disease causing)
- Do the parents have the same change (parent is fine and has the CNV = less likely to be causative, consider penetrance)
26
What is Di George syndrome?
Caused by deletion of the TBX1 gene at 22q11.2
27
What are incidental findings? (2)
- A genetic problem that you aren't looking for but couldn't avoid finding
- Unexpected and unrelated to the referral reason, may have severe implications for the patient and relatives
28
What is penetrance? (3)
- A measure of the likelihood of a genetic variant causing a disease
- A variant/CNV doesn't always cause medical problems
- Expressed as a percentage of times a variant would be expected to cause a disease (100% penetrance = 100% of people with the variant have the disease)
29
What does genome sequencing achieve? (4)
- Detect single nucleotide DNA variants
- With bioinformatics can detect copy number and balanced rearrangements
- Mosaicism is dependent on depth of coverage
- But generates lots of data
30
What does karyotype and FISH achieve? (3)
- Large abnormalities
- Balanced rearrangements
- 10% mosaicism detection
31
What do microarrays achieve? (3)
- Smaller abnormalities than karyotype and FISH because more sensitive
- Can't detect balanced changes
- 10-30% mosaicism detection
32
What is quantitative fluorescent PCR used for? (3)
- Detecting aneuploidy in prenatal testing of amniotic fluid and chorionic villus samples at risk of Down syndrome (copy number assay)
- Urgent 24 hour test
- Amplifies and quantifies at the same time
33
How does QF-PCR work? (7)
- Uses fluorescently labelled PCR primers which are targeted to amplify small tandem repeats (STRs)
- STRs are repeats of two or more nucleotides, number of repeats varies between people, for an STR on chromosome 21 one allele may have 5 repeats the other allele may have 9
- Both alleles are amplified
- Disomic (no gain): both alleles should amplify to the same degree so same number of DNA fragments for each allele
- Relative copy number is measured by fluorescence intensity
- Disomic: 2 peaks of similar size (1:1)
- Trisomy: 3 peaks (1:1:1) each with different size STRs or 2 peaks (2:1) if 2 alleles have same STR size
34
What is digital droplet PCR? (7)
- Used to test for a specific DNA variant to a higher resolution (e.g. if only present in 1 out of 20 000 cells) to identify low level mosaicism
- Separate patient DNA into separate droplets and do PCR reactions
- A portion of the droplets will contain the variant of interest and a portion are wildtype
- If patient is heterozygous, 50% of droplets will have the variant of interest
- 2 DNA probes are used (WT or variant) with a fluorescent marker and quencher molecule (which prevents fluorescence)
- If probe binds, quencher is separated so the fluorescence is emitted and registered by scanner so each droplet is positive for variant or WT fluorescence
- Single cell analysis but look at 1000s of cells
35
How do you interpret ddPCR output? (3)
- Four clusters of droplets
- Double negative (no targeted DNA templates), WT only, mutant only, double positive (contain both WT and mutant templates)
- Can see proportion of cells with the variant so good technique for exclusion and measurement of mosaicism
