Lecture 12: Molecular Cytogenetics Flashcards
What is a copy number variant?
A gain or loss of genetic material
CNVs are a _________ source of variation and makes up ______% of the genome. They can range from __bp to ___bp
Major Source
5-10%
50bp - 3Mbp
CNVs can be the loss or gain of the following:
- Whole Chromosomes
- Several Adjacent genes in a contiguous gene syndrome
- Single Gene
- Exons
What are the methods of CNV detection?
Karyotyping FISH QF-PCR DNA microarray MLPA NGS
What is QF-PCR and what is it used for?
Quantitative Fluorescent PCR, prenatal diagnosis of aneuploidies
What aneuploidies are compatible with life?
13 (Patau Syndrome), 18(Edwards Syndrome), 21 (Down Syndrome), X and Y
How do we get the tissue for QF-PCR?
Invasive sampling of Amniotic fluid or Chorionic Villus Sampling, foetal DNA is obtained and short tandem repeat fluorescent markers are used to detect aneuploidies
How do we interpret the results of QF-PCR?
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
What are the advantages of QF-PCR?
- Robust
- Rapid 24-48 hours
- Cost effective
- Can be automated
What is NIPT and what does it test?
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
What is a DNA microarray?
A DNA chip covered in unique oligonucleotide probes that are used as hybridisation targets, fluorescence is used to detect the hybridisation and quantified
What are the 2 different types of microarray?
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)
Describe the Illumina DNA microarray:
- 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
What is the Log R Ratio (LRR)?
The normalised measure of the total signal intensity for a SNP in the SNP assay. Any deviation from 0 = CNV
What is the B Allele Frequency (BAF)?
A measure of the allelic intensity ratio, the proportion of the sample that carries the B allele as designated by the Infinium assay
What is the LRR for a deletion?
LRR is negative
What is the LRR for a duplication?
LRR is positive
What do Regions of Homozygosity (ROH) suggest?
- Uniparental disomy (Imprinted regions)
- Identity by descent (Close ancestry/isolated ethnic population)
What are the advantages of DNA microarray over Karyotyping?
- Higher resolution and therefore diagnostic yield
- Cheaper because it can be automated
- Robust technology
- Tissue culture isn’t needed
What are the disadvantages of DNA microarray?
- 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)
What is MLPA?
Multiplex Ligation-dependent Probe Amplification is a test that is targeted to specific regions of interest and not the whole genome
What is the MLPA procedure?
- Denaturation of the target DNA and Hybridisation of 2 oligo probes
- Ligation of the 2 probes
- PCR using universal primers
- Electrophoresis and analysis of the PCR products
What are the pros and cons of NGS?
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
Give an example of a disease caused by a whole chromosome CNV:
Down Syndrome
- Mental retardation, dysmorphic features
- increased risk of heart problems
What tests are done for whole chromosome CNVs?
QF-PCR first
NIPT
FISH if mosaicism is suspected
Karyotyping
How are several adjacent genes in a contiguous gene syndrome causing CNVs detected?
Usually microarray, MLPA or FISH
Give an example of a disease caused by adjacent gene CNVs:
DiGeorge syndrome - have a 22q11.2 deletion - CATCH 22 Cardiac abnormalities Abnormal Faces Thymic Hypoplasia/Underdeveloped immunity Cleft palate Hypocalcaemia
Give an example of a disease caused by a single gene CNV:
Charcot Marie Tooth disease:
- caused by a gain of PMP22
- progressive adult onset peripheral neuropathy
- can be detected by MLPA and FISH
How are CNVs involving exons detected?
MLPA
