Microarrays in genomics

Question 1

What is a constitutional genetic disorder? (2)

Answer 1

• Genetic aberrations which are present in all cells from birth (also called germline)
• Somatic genetic aberrations are acquired later in life and are mosaic

Question 2

What is the resolution of karyotyping?

Answer 2

5-20 megabase

Question 3

What is the principle of karyotyping? (3)

Answer 3

• Trypsin digestion produces light and dark bands
• Dark = AT rich, heterochromatin
• Light = GC rich, euchromatin

Question 4

How does FISH work? (6)

Answer 4

• 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

Question 5

What are the types of FISH probe? (2)

Answer 5

• Whole chromosome paint
• Locus specific probe

Question 6

What can FISH show? (3)

Answer 6

• Gene amplification
• Translocations
• Gene fusion

Question 7

What is a microarray? (5)

Answer 7

• 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

Question 8

What is the benefit of karyotyping over microarrays?

Answer 8

Microarrays can’t detect genetically balanced rearrangements e.g. translocations

Question 9

Which patients are tested using microarrays? (3)

Answer 9

• 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)

Question 10

What are the benefits of microarrays over karyotyping? (3)

Answer 10

• 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

Question 11

What are the 2 main types of microarrays?

Answer 11

• Oligonucleotide arrays
• Single nucleotide polymorphism (SNP) arrays

Question 12

What are oligonucleotide arrays?

Answer 12

Uses oligonucleotide DNA probes that are complimentary to specific regions of the genome

Question 13

What are SNP arrays?

Answer 13

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

Question 14

How do oligonucleotide arrays work? (7)

Answer 14

• 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

Question 15

What do oligonucleotide arrays detect? (2)

Answer 15

• Copy number variants (CNVs)
• Do not detect genetically balanced rearrangements e.g. translocations and inversions

Question 16

When are apparent losses/gains significant on an oligonucleotide array profile? (2)

Answer 16

• At least 3 neighbouring probes showing a loss or gain
• Single calls are likely to be artefacts

Question 17

What is an SNP? (6)

Answer 17

• 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)

Question 18

How do SNP arrays work? (9)

Answer 18

• 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

Question 19

What is loss of heterozygosity? (4)

Answer 19

• 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

Question 20

How do you interpret the data from SNP array? (5)

Answer 20

• 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

Question 21

What does a deletion look like on a SNP array? (3)

Answer 21

• 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

Question 22

What does a duplication look like on a SNP array? (3)

Answer 22

• 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

Question 23

What is Pallister-Killian syndrome? (7)

Answer 23

• 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

Question 24

Is a gain or loss more likely to have a phenotypic consequence?

Answer 24

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)

Question 25

How should a CNV be assessed? (6)

Answer 25

• 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)

Question 26

What is Di George syndrome?

Answer 26

Caused by deletion of the TBX1 gene at 22q11.2

Question 27

What are incidental findings? (2)

Answer 27

• 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

Question 28

What is penetrance? (3)

Answer 28

• 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)

Question 29

What does genome sequencing achieve? (4)

Answer 29

• 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

Question 30

What does karyotype and FISH achieve? (3)

Answer 30

• Large abnormalities
• Balanced rearrangements
• 10% mosaicism detection

Question 31

What do microarrays achieve? (3)

Answer 31

• Smaller abnormalities than karyotype and FISH because more sensitive
• Can’t detect balanced changes
• 10-30% mosaicism detection

Question 32

What is quantitative fluorescent PCR used for? (3)

Answer 32

• 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

Question 33

How does QF-PCR work? (7)

Answer 33

• 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

Question 34

What is digital droplet PCR? (7)

Answer 34

• 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

Question 35

How do you interpret ddPCR output? (3)

Answer 35

• 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