Lecture 12: Molecular Cytogenetics

Question 1

What is a copy number variant?

Answer 1

A gain or loss of genetic material

Question 2

CNVs are a _________ source of variation and makes up ______% of the genome. They can range from __bp to ___bp

Answer 2

Major Source
5-10%
50bp - 3Mbp

Question 3

CNVs can be the loss or gain of the following:

Answer 3

• Whole Chromosomes
• Several Adjacent genes in a contiguous gene syndrome
• Single Gene
• Exons

Question 4

What are the methods of CNV detection?

Answer 4

Karyotyping
FISH
QF-PCR
DNA microarray
MLPA
NGS

Question 5

What is QF-PCR and what is it used for?

Answer 5

Quantitative Fluorescent PCR, prenatal diagnosis of aneuploidies

Question 6

What aneuploidies are compatible with life?

Answer 6

13 (Patau Syndrome), 18(Edwards Syndrome), 21 (Down Syndrome), X and Y

Question 7

How do we get the tissue for QF-PCR?

Answer 7

Invasive sampling of Amniotic fluid or Chorionic Villus Sampling, foetal DNA is obtained and short tandem repeat fluorescent markers are used to detect aneuploidies

Question 8

How do we interpret the results of QF-PCR?

Answer 8

Diploid = allele peaks in a ratio of 1:1
Trisomy = allele peaks in a ratio of 2:1 if 2 alleles and 1:1:1 if 3 alleles

Question 9

What are the advantages of QF-PCR?

Answer 9

• Robust
• Rapid 24-48 hours
• Cost effective
• Can be automated

Question 10

What is NIPT and what does it test?

Answer 10

Non-Invasive Prenatal Testing tests foetal DNA in the blood for aneuploidies. Done after 10 weeks of gestation but is not diagnostic, just a screening test

Question 11

What is a DNA microarray?

Answer 11

A DNA chip covered in unique oligonucleotide probes that are used as hybridisation targets, fluorescence is used to detect the hybridisation and quantified

Question 12

What are the 2 different types of microarray?

Answer 12

Array CGH:
- tests genomic gains or losses
SNP array:
- tests genomic gains or losses
- also tests copy neutral aberrations like regions of homozygosity (ROH) and loss of heterozygosity (LOH)

Question 13

Describe the Illumina DNA microarray:

Answer 13

• Each bead is covered with hundreds of thousands of copies of specific oligos
• Oligos bind to complementary sequences in the DNA
• Fluorescence excitation by scanners and the signal capture results in intensity calculation
• Allele ratios can then be calculated

Question 14

What is the Log R Ratio (LRR)?

Answer 14

The normalised measure of the total signal intensity for a SNP in the SNP assay. Any deviation from 0 = CNV

Question 15

What is the B Allele Frequency (BAF)?

Answer 15

A measure of the allelic intensity ratio, the proportion of the sample that carries the B allele as designated by the Infinium assay

Question 16

What is the LRR for a deletion?

Answer 16

LRR is negative

Question 17

What is the LRR for a duplication?

Answer 17

LRR is positive

Question 18

What do Regions of Homozygosity (ROH) suggest?

Answer 18

• Uniparental disomy (Imprinted regions)

- Identity by descent (Close ancestry/isolated ethnic population)

Question 19

What are the advantages of DNA microarray over Karyotyping?

Answer 19

• Higher resolution and therefore diagnostic yield
• Cheaper because it can be automated
• Robust technology
• Tissue culture isn’t needed

Question 20

What are the disadvantages of DNA microarray?

Answer 20

• Unable to detect balanced rearrangements
• No positional information for a duplication
• Time-consuming to analyse and report
• Many CNVs are novel with no clinical significance
• Can detect things you weren’t planning on detected (Tumor suppressor gene deletion etc)

Question 21

What is MLPA?

Answer 21

Multiplex Ligation-dependent Probe Amplification is a test that is targeted to specific regions of interest and not the whole genome

Question 22

What is the MLPA procedure?

Answer 22

1. Denaturation of the target DNA and Hybridisation of 2 oligo probes
2. Ligation of the 2 probes
3. PCR using universal primers
4. Electrophoresis and analysis of the PCR products

Question 23

What are the pros and cons of NGS?

Answer 23

Pros:
- Higher resolution of CNVs
- Breakpoint analysis
- Prediction of absolute copy number arrays
Cons:
- Many algorithms can't detect CNVs
- Expensive
- Time-consuming
- Too much data to store

Question 24

Give an example of a disease caused by a whole chromosome CNV:

Answer 24

Down Syndrome

• Mental retardation, dysmorphic features
• increased risk of heart problems

Question 25

What tests are done for whole chromosome CNVs?

Answer 25

QF-PCR first
NIPT
FISH if mosaicism is suspected
Karyotyping

Question 26

How are several adjacent genes in a contiguous gene syndrome causing CNVs detected?

Answer 26

Usually microarray, MLPA or FISH

Question 27

Give an example of a disease caused by adjacent gene CNVs:

Answer 27

DiGeorge syndrome 
- have a 22q11.2 deletion
- CATCH 22 
Cardiac abnormalities
Abnormal Faces
Thymic Hypoplasia/Underdeveloped immunity
Cleft palate
Hypocalcaemia

Question 28

Give an example of a disease caused by a single gene CNV:

Answer 28

Charcot Marie Tooth disease:

• caused by a gain of PMP22
• progressive adult onset peripheral neuropathy
• can be detected by MLPA and FISH

Question 29

How are CNVs involving exons detected?

Answer 29

MLPA