Using DNA sequencing to diagnose single gene disorders Flashcards
What are the types of genetic variants?
- Deletion
- Insertion
- Substitution - One base substituted for another
Describe substitution
Missense - Where a change in nucleotide base causes that codon to code for an entirely different amino acid. This can completely change the structure and function of a protein
Nonsense - Variants causing a premature stop codon to be formed
Describe insertion/deletion
- Can be frameshift variants
- If you have a deletion or insertion divisible by 3 it will result in an in-frame deletion i.e. the amino acid sequence after the insertion/deletion will remain unchanged
- If the number of nucleotides is not divisible by 3, then ‘out of frame variation’ occurs, and every codon downstream changes - This is frameshift, and has a much more significant functional impact
Which genetic variants are most and least likely to cause disease?
Pathogenicity - Likelihood to lead to disease
Most likely to least likely:
- Synonymous - Not predicted to change sequence of protein, unlikely to cause problems
- Missense - Can causes problems depending on location
- Null/Nonsense - Likely to be clinically significant/pathogenic
How are variants classified?
- ACMG/AMP guidelines:
- 1 or 2 (<5%)= Benign, likely benign
- Pathogenic is only labelled on a variant when there’s 95% certainity it causes disease
- Anything in b/w these 2 values is of unknown/uncertain significance
- Specific guidelines are used to classify variants
- Uses a series of specific criteria to assign levels fo evidence for pathogenicity or benignty
Describe PCR
- Replicates/copies DNA on a large scale
- Reaction components are:
- Template DNA
- Forward and reverse primers
- dNTPs
- DNA polymerase
- MgCl2 (enzyme cofactor)
- Buffer and water
- Each cycle:
- Denaturation - 94-98 degrees
- DNA strands separated
- Annealing - 50 - 65 degrees
- Add primers which bind to complementary sites one each strand
- Extension - 72 degrees
- DNA Taq polymerase binds to the primers and fills in the gap in each direction with complementary nucleotides dNTP
- Denaturation - 94-98 degrees
Describe Sanger sequencing
- Primary gold standard for DNA sequencing
1 - Denature dsDNA using heat
2- Make multiple copies of segment
3- Attach primer
4 - Add to 4 polymerase solutions
5 - Grow complementary chains until termination dye
6 - Denaturate the grown chains
7 - Electrophorese the 4 solutions
- Has primer and polymerase but in addition to doxynucleotides, have dideoxynucleotides (lost an oxygen molecule from ribose ring) - This means it cannot extend th esequence chain any further
- The 4 types of ddNTP are bound to different colour probes
- Target DNA is copied many times, makes fragments that differ in length by a single nucleotide
- As the strands are all different lengths, they can be separated using a gel or capillary electrophoresis on a sequencer and it tells you at each strand legth what base is preent, according to its fluorescence peak
Describe Next Generation Sequencing
- Collection of technologies enabling massively parallel sequencing
- Sequencing of hundreds of thousands of fragments of DNA at the same time
Steps in NGS pathway:
- Library Preparation: DNA samples are fragmented and a custom adapter sequence added
- Enrichment: Pulling out the region of interest for sequencing
- Amplification: The library fragment is samplified to produce DNA clusters
- Sequencing: Each DNA cluster is simultaneously sequenced and that data captured
- Alignment/mapping: Read sequences (’reads’) are compared to a reference template
- Variant calling: Variants are identified as differences b/w the read and the reference genome
- Variant annotation: The likely effect that a genetic variant will have on a protein is identified
- Variant interpretation
Compare single gene sequencing, gene panel sequencing, exome sequencing and whole genome sequencing
Single gene - 1 gene
Gene panel - 2 → hundreds of genes
Whole exome - 20,000 genes (but only coding sequence)
Whole genome - Entire genome (including non-coding regions)
What are the pros and cons of single gene testing?
Pros:
- Quick
- Cheap
- Reliable
- Low risk of incidental findings and/or VUS
Cons:
- Miss non-coding variants
- Miss dosage abnormalities
- Miss mosaicism
What are the pros and cons of gene panel sequencing?
Pros:
- Multiple genes
- Fast
- Cost effective
- Good read depth (e.g. can detect mosaicism)
Cons:
- Limited to genes selected for panel
- Difficult to update
- Does not detect structural rearrangements
- Misses non-coding regions
What are the pros and cons of whole exome sequencing?
Pros:
- Can identify novel gene-disease associations
- No need to update (can choose genes to analyse)
- Can be performed rapidly if indicated (e.g. prenatal)
Cons:
- Limited read depth/patchy coverage
- Does not (usually) detect structural rearrangements
- Risk of VUS and incidental findings
- Misses non-coding regions
What are the pros and cons of whole genome sequencing?
Pros:
- Can identify novel gene-disease associations
- Uniform coverage (including non-coding regions)
- Can detect structural rearrangements and dosage abnormalities
- Option to apply virtual panels and reanalyse data
Cons:
- Costly
- Time consuming
- Poorer read depth
- High risk of VUS/ incidental findings
- Large data volume → Difficult interpretation
What’s the threshold of certainity needed to have to say a variant is disease causing before you can change clinical management?
> 90%
When is single gene sequencing used?
When a patient’s features are strongly indicative of a genetic condition caused by variants in a single gene
When is whole exome sequencing used?
To identify novel genetic causes of disease
When is whole genome sequencing used?
Increasingly used in patients with cancer, where the genome of the tumour can be sequenced to look for mutational burdens and driver mutations
