Technologies Flashcards
(16 cards)
How does the technology fo Arrays work?
An array with immobilised allele-specific oligonucleotide (ASO) probes
Fragmented nucleic acid sequences from the target sample, labelled with fluorescent dyes
A detection system that records and interprets the hybridisation signal
How it Works: Fragmented sample DNA hybridises to complementary probes on the array. A single base extension step incorporates a fluorescently labelled nucleotide. This nucleotide emits a signal when excited by a laser
Signal Intensity:
Depends on the amount of target DNA in the sample (used to detect copy number)
Depends on the affinity between target DNA and probe (binding is more efficient with 100% matched bases than with mismatches due to a SNP)
Data Conversion: Computational algorithms convert probe signal intensity into genotypes
What does SNP arrays detect?
Array data on the presence/absence of more than one allele is used for detecting UPD and LOH. Combined information on the number of alleles at individual SNP sites is used for determining copy number.
What is the Log R Ratio Chart and what does it mean and what is the graph like in UPD?
-The Log R chart is used to interpret copy number changes
A positive shift in the Log R ratio indicates a copy number gain (duplication or amplification)
A negative shift indicates a copy number loss (deletion or nullisomy)
The Log R ratio typically does not show copy number changes in cases of Uniparental Disomy (UPD) or Copy Number Neutral LOH (AOH)
What is the B-Allele Frequency (BAF) Chart Theory?
*The B-Allele Chart shows the proportion of the “B” allele for each SNP
*For each SNP, one allele is designated “A” and the other “B”
*The B-allele frequency is calculated as: Number of B alleles / (Number of A alleles + Number of B alleles)
*Expected Values in a Normal Diploid (2n) Individual: When plotted in order along the chromosome, BAF values typically cluster at three points, representing different genotypes
◦AA Homozygotes: BAF = 0 / (2+0) = 0.0
◦BB Homozygotes: BAF = 2 / (0+2) = 1.0
◦AB Heterozygotes: BAF = 1 / (1+1) = 0.5
Centromeres typically appear as a gap in the BAF chart
What is the Duplication (Gain) BAF pattern?
If there is a gain, heterozygous SNPs (normally at 0.5) will now have three copies (e.g., AAB or ABB)
AAB configuration: BAF = 1 / (2+1) = 0.333
ABB configuration: BAF = 2 / (1+2) = 0.666
The single cluster at 0.5 splits into two clusters at 0.333 and 0.666
What is the deletion (loss) BAF pattern?
If there is a deletion, there will be only one copy of each region, meaning no heterozygous SNPs
Only A allele present: BAF = 0 / (1+0) = 0.0
Only B allele present: BAF = 1 / (0+1) = 1.0
All SNPs cluster at 0.0 or 1.0, with none at 0.5 (Note: This pattern also looks like Isodisomy UPD or AOH).
What is the BAF pattern for mosaic dels and dups?
Mosaic deletions: Cause a shift from the expected 0.0 (AA) and 1.0 (BB) clusters towards the middle (0.5). A larger shift indicates more “normal cells”
Mosaic duplications: Cause a shift from the expected 0.333 (AAB) and 0.666 (ABB) clusters towards the middle (0.5)
. A larger shift towards 0.5 indicates more “normal cells”
Mosaic trisomy (extra copy different from original two) can show six BAF lines
Detection Reliability: Generally not reliable below 20-30%, especially for small clones or minimal residual disease (MRD)
. Higher resolution arrays may improve this, but NGS or digital PCR are typically preferred for low-level detection
What is the BAF pattern in UPD?
Definition: Occurs when both copies of a chromosome are inherited from the same parent
Crucially, the Log R ratio does not indicate a copy number change in UPD
What is the BAF pattern in isodisomy?
Both inherited copies are identical
Every SNP will be homozygous (AA or BB)
The BAF chart will show only AA (0.0) and BB (1.0) clusters along the affected chromosome
This pattern looks like a deletion on the BAF chart but without the LogR loss
Indicates autozygosity or loss of heterozygosity (LOH). Also known as AOH (Absence of Heterozygosity)
What is heterodisomy?
Two different homologues from the same parent are inherited
◦Detected by SNP arrays, especially with a trio (mother, father, child) for comparison
◦Shows a mixture of AA (0.0), AB (0.5), and BB (1.0) alleles along the affected chromosome. This reflects inheriting two non-identical copies from one parent
◦Software analysis with a trio can provide a Mendelian inheritance score to detect heterodisomy UPD
*Example (Prader-Willi Syndrome): Maternal UPD of chromosome 15 leading to Prader-Willi syndrome can show a mixture of AA, AB, and BB alleles (heterodisomy) or solely AA and BB (isodisomy)
. Large areas of heteroisodisomy (mix of patterns) can also be seen
.Clinical Significance: UPD can be linked to conditions like Prader-Willi or Angelman syndrom
What is Loss of Heterozygosity (LOH) / Absence of Heterozygosity (AOH)
Occurs when there are no copy number gains or losses, but heterozygosity is lost at specific loci
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Detection: Detected by SNP arrays which identify regions where heterozygosity is lost and all SNPs are homozygous (AA or BB)
BAF Pattern: The BAF chart shows a lack of heterozygous (0.5) SNPs in the region, clustering only at 0.0 and 1.0
Log R Ratio: The LogR ratio remains normal as there is no gain or loss of material
. This is the key distinction from a true deletion.
What is the significance of AOH/LOH?
Can indicate genetic relatedness, such as consanguinity, where regions are identical by descent (IBD), leading to more homozygous alleles
. Significant LOH across multiple chromosomes suggests consanguinity. Clinical policy is needed for reporting such findings sensitively
Relevant for identifying recessive disorders (as homozygous regions may unmask recessive mutations)
and imprinting disorders (like Prader-Willi or Angelman syndrome). Particularly useful in malignancy arrays to identify tumour suppressor gene loss and other alterations characteristic of cancer
What is the main difference beterrn SNP and oligo array?
SNPs do not occur evenly across the genome, leading to uneven coverage on SNP-only arrays
Solution: Combined arrays add standard oligonucleotide probes to the SNP design
This helps fill in the gaps and improve genomic coverage
Outcome: Leads to more comprehensive and reliable copy number detection
What is the application of SNP arrays?
First-line test for patients with developmental delay and/or dysmorphism in many UK labs
Can help unmask potential recessive diseases in families with suspected inheritance by identifying long stretches of homozygosity (further testing needed)
What is the application of SNP arrays in oncology?
LOH and Allelic Imbalance (AI): Widely used to identify AI, helping refine cancer subtype classification (e.g., lung cancer types). Crucial for studying tumour heterogeneity and can be a marker for treatment response and identifying inactivated tumour suppressor genes
◦DNA Copy Number Aberration (CNA) Analysis: Essential for detecting CNAs (e.g., 8q gain in prostate cancer)
. Crucial for identifying CNVs associated with cancer progression, metastasis, and personalised therapeutic strategies
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◦Combined LOH and DNA Copy Number Analysis: Better than SNP array alone
. Pioneered studying LOH without DNA copy number loss in malignancies. Example: detecting UPD regions coinciding with homozygous mutations in AML
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Methylation Analysis: Widely adopted for epigenetic analysis
. Established approach for detecting DNA methylation patterns indicative of cancer prognosis/diagnosis. Newer platforms are specifically designed for epigenetic studies. Principle can involve restriction enzyme digestion and comparison
Allelic Specific Gene Expression: Can be detected using cDNA as starting material; may be important in tumourigenesis
What are the limitations of SNP Arrays
Cannot detect balanced rearrangements (translocations, inversions), gene fusions, or whole genome ploidy changes
Better detected by: Next-generation sequencing (NGS) or chromosome-based methods like FISH or aCGH
SNP arrays are not ideal for detecting whole genome ploidy changes because they don’t reliably identify balanced or subtle structural variants
Mosaicism Detection: Generally not reliable for detecting mosaicism below 20-30%
. Less suitable for minimal residual disease (MRD) detection
Better detected by: NGS or digital PCR for higher sensitivity, especially for MRD or specific nucleotide mutations
. High-resolution SNP arrays can improve detection sensitivity
Coverage: SNPs do not occur evenly, leading to uneven genomic coverage unless combined with oligo probes
