12 - Molecular Cytogenetics 1 Flashcards
(27 cards)
Structural Variation (SV)
- Rearrangements of the DNA in a genome resulting in novel breakpoint junctional events
- Copy number neutral (Inversion or balanced translocation)
- Copy number variants (Deletion, duplication, triplication)
Copy number variants
- Major source of genetic diversity
- Common
- Mostly benign polymorphic structural
variation (SV) with no phenotypic effect - Can have a role in human disease (e.g. obesity and cancer predisposition
Role of genomic architecture on human genetic disease
Structural features of the human genome or the genomic architecture, can result in region-specific susceptibility to rearrangements and thus genomic instability which can result in human genetic disease.
Genomic disorders
Loss or gain of:
- Whole chromosome (aneuploidy)
- Several adjacent genes in a contiguous gene syndrome
(microdeletion/microduplication syndrome)
- Single gene
- Exons (part of a gene)
Molecular mechanisms through which phenotypes of genomic disorder can arise
- Gene dosage (altering copy number of a dosage sensitive gene, e.g haploinsufficiency)
- Gene interruption (exon deletion)
- Gene fusion
- Position effect
- Deletion unmasking recessive variant on the other allele
Methods of detecting CNVs
- Karyotype
- FISH (metaphase & interphase)
- QF-PCR
- DNA microarray
- MLPA
- NGS
QF-PCR
- Prenatal diagnosis of abnormal chromosome copy number (aneuploidies)
- Only a few aneuploidies are compatible with life (13, 18, 21, X and Y)
- At risk pregnant woman undergoes invasive sampling of amniotic fluid or CVS (foetal DNA is obtained)
- Performed using multiplex of fluorescent, polymorphic STR (short tandem repeat) markers from chromosomes 13, 18, 21, X and Y
- Copy number is determined from the relative quantification of STR markers (normal if peak 1:1, trisomy = 1:1:1)
Advantages of QF-PCR
- Accurate
- Low failure rate
- Rapid
- Cost effective
- Suitable to automation
What does QF PCR stand for
Quantitative Fluorescent PCR
DNA microarray
- DNA chips
- Whole genome analysis
- A collection of DNA spots attached to a solid surface
- Each DNA spot contains a specific DNA sequence, known as a probe (oligonucleotide)
- Probes are used as hybridisation targets
- Probe-target hybridization is usually detected and quantified by detection of fluorophore
Advantages of microarray compared to karyotyping
- Much higher resolution and therefore diagnostic yield
- Cheaper
- Robust
- Tissue culture not needed
Disadvantages of microarray
- Unable to detect balanced rearrangements
- No positional information for a duplication
- Can be time consuming to analyse and report
- Many CNVs are novel with unknown clinical significance
- Can detect incidental findings (eg deletion of cancer suppressor genes)
Microarray uses
- Prenatal diagnosis
- Pregnancy loss
- Cancer
Types of chromosome microarrays
- Array CGH (Genomic gains and losses)
- SNP array (genomic gains and losses plus copy neutral aberrations)
Array CGH
- Patient genomic DNA compared to normal reference control DNA
- Fragment DNA, fluorescently labelled in different colours and then competitively hybridised on to the array and read with a fluorescence scanner.
SNP arrays
- Do not use control reference DNA
- Fragmented patient DNA is hybridised to the array
- SNP allele frequency and absolute fluorescence levels are compared to a standard consisting of averaged results for multiple normal samples
Detection of CNVs
Log R Ratio and B Allele Frequency
Log R Ratio (LRR)
- Log R Ratio (LRR) is a normalised measure of the total signal intensity for the SNP
- Any deviations from zero for LRR is evidence for copy number change.
B Allele Frequency (BAF)
Measure of the allelic intensity ratio
Diagnostic implications regions of homozygosity (ROH)
- Uniparental disomy (single large ROH on same chromosome)
- Identity by descent (multiple ROH across different chromosomes)
5 tiers to classification of CNV
- Pathogenic
- Likely pathogenic
- Uncertain
- Likely benign
- Benign
Reporting pathogenic / likely pathogenic microarray results
- Testing family members may be appropriate.
- Test parents for recurrence risk.
- Rule out balanced rearrangement
- Genetic counselling recommended
Reporting CNV uncertain microarray results
- Testing parents may be helpful
- Genetic counselling may be appropriate
Multiplex Ligation dependent Probe Amplification (MLPA)
- Targeted to specific regions of interest NOT whole genome analysis
- Have probes that target a specific genome sequence
- MLPA probe consists of two parts (left and right probe oligonucleotide - LPO, RPO)
