Clinical Genetics: Overview of genomic technologies in clinical diagnostics Flashcards
1
Q
Give a brief overview of what PCR (polymerase chain reaction) is and how it works
A
- PCR is used to amplify a specific region of DNA, e.g. a gene or gene exon associated with a disease
- 3 stages: Denaturation, annealing and extension
- Each cycle doubles the amount of DNA copies of your target sequence
- Amplify enough DNA molecules so that we have sufficient material for downstream applications, e.g. sanger sequencing
2
Q
What is fragment analysis?
A
- PCR followed by capillary electrophoresis which sizes the PCR product accurately
- Instead of being represented by bands as with gel electrophoresis the PCR products are represented by peaks
3
Q
What can fragment analysis be used for?
A
- Can be used to detect repeat expansions or other small size changes in allele size (up to a few hundred bp)
- This is important because repeat expansions can cause diseases such as Huntingdon’s disease
4
Q
What is Huntingdon’s disease?
A
- It is a severe neurodegenerative disorder caused by CAG repeat expansion in the Huntingtin (HTT) gene
5
Q
How does the CAG expansion in the Huntingtin gene causes Huntingdon’s disease?
A
- Normal HTT gene has < 27 CAG copies; Intermediate 27-35 copies; Pathogenic > 35 copies
- Expanded protein produced from HTT gene with CAG repeat expansion is toxic and accumulates in neurons causing cell death
6
Q
How is Huntingdon’s disease diagnosed?
A
- Diagnosed with fragment analysis
7
Q
Give a brief overview of sanger sequencing
A
- Cycle Sequencing technique; based on the same principles as PCR
- Used to re-construct a nucleotide sequence
- Each of the 4 dideoxyribose nucleoside triphosphates (ddNTPs) in reaction mixture has a different dye so we can determine the nucleotide sequence.
- Can sequence up to 800bp of sequence per reaction so good for sequencing single exons of genes
8
Q
What are some of the disadvantages of sanger sequencing?
A
- Slow
- Low-throughput
- Costly to perform for large numbers of samples
9
Q
What can sanger sequencing be used for?
A
- Can be used to identify single nucleotide polymorphisms (SNPs), or mutations
10
Q
Give a brief overview of Fluoresence in situ hybridisation (FISH)
A
- Used to microscopically detect large chromosomal abnormalities such as:
- Extra chromosomes
- Large deleted segments
- Translocations
11
Q
How does fluoresence in situ hybridisation work?
A
- Design Fluorescent probe to a chromosomal region of interest
- Denature probe and target DNA
- Mix probe and target DNA together (hybridisation)
- Probe binds to the target DNA on the chromosome of interest
- Target fluoresces or lights up
12
Q
Give a brief overview of Array CGH (comparative genomic hybridisation)
A
- Used for detection of sub-microscopic chromosomal abnormalities
- Patient DNA labelled Green
- Control DNA labelled Red
13
Q
How does Array CGH work?
A
- Patient DNA and control DNA are extracted from samples
- Patient DNA labelled with Cy3, green and control DNA labelled with Cy5, red
- They are then mixed together and hybridised to the microarray
- Patient and control DNA compete to hybridise to the microarray
- Each spot on the array is then scanned to identify the colour of fluoresence it produces
14
Q
What does each colour of fluoresence on the microarray for array CGH represent?
A
- No colour fluoresence = Equal hybridisation of patient and control DNA
- Red = More control DNA hybridised than patient DNA so there’s a loss of DNA in patient in that position in genome
- Green = More control DNA hybridised than control DNA so there’s a gain of DNA in patient in that position in genome
15
Q
What is MLPA?
A
- Multiplex ligation-dependent probe amplification (MLPA)
- It’s a variation of PCR that permits amplification of multiple targets
- MLPA is used to detect abnormal copy numbers at specific chromosomal locations
- Can also detect sub-microscopic (small) gene deletions/partial gene deletions
- Usually used to see if there’s any abnormalities in a gene known to be involved in a specific condition
16
Q
How does MLPA work?
A
- Each probe consists of two oligonucleotides which recognize adjacent target sites on the DNA
- One oligonucleotide probe contains the sequence recognized by the forward primer,
- The other oligonucleotide probe contains the sequence recognized by the reverse primer.
- Only when both probe oligonucleotides are hybridized to their respective targets, can they be ligated into a complete probe
- DNA that’s bound to complete probe is then amplified
- Perform fragment analysis (capillary electrophoresis) on the MLPA product
17
Q
Why is MLPA an example of a quantitative assay?
A
- Because the amount of MLPA product produced is proportional to the amount of starting DNA
18
Q
How is the data from MLPA analysed?
A
- MLPA product/s produce peaks which represent the no. of copies of a chromosome at that particular area of the genome
- The signal strengths of the probes are compared with those obtained from a reference DNA sample known to have two copies of the chromosome
19
Q
Why has next generation sequencing replaced sanger sequencing for almost all sequencing tests?
A
- Higher DNA sequencing throughput
- Provides a wider range of tests in a shorter time for less money
20
Q
Why is whole exome sequencing used more often tha whole genome sequencing?
A
- 80% pathogenic mutations are protein coding
- Therefore it’s more efficient and cheaper to only sequence the exome if we want to locate a pathogenic mutation within the genome
21
Q
How does whole exome sequencing work?
A
- Use a process called Target enrichment
- Take DNA frgaments and hybridise them with RNA baits that are complementary to the exons of the DNA fragments
- Once hybridisation occurs the bait-bound DNA fragments will be “captured” using streptavidin coated beads
- These beads are magnetic so using a magnet will separate the fragments containing exon sequences from the fragments that don’t contain them
- Beads are then washed away and the RNA baits are digested leaving only the DNA fragments containing exons
- Then perform next generation sequencing as normal
22
Q
It’s Universally accepted that genome sequencing will become commonplace in diagnostic genetics but not all tests will move to WGS, what tests won’t be replaced by WGS?
A
- Panels/single gene tests may still be more suitable for some diseases, e.g. cystic fibrosis
- Capillary-based methods: Repeat expansions, MLPA, family mutation confirmation Sanger sequencing
- Array-CGH: large sized chromosomal aberrations
23
Q
What is the main disadvantage of Whole genome sequencing currently?
A
- Data generation and data procesing is an automated process in whole genome sequencing
- However, whole genome sequencing still requires manual interpretation which is ver time consuming and challenging
24
Q
What are some other disadvantages of whole genome sequencing?
A
- Ethical considerations
- Modified patient consent process needed
- May also find defetcs/mutations that put future family at risk of a specific disease so strategy for reporting ‘incidental’ findings needs to be produced
- Infrastructure and training (particularly IT and clinical scientists) needs to be put in place
25
What is the NHS Diagnostic Laboratory?
* Accredited laboratory that:
* Provide clinical and laboratory diagnosis for genetic disorders
* Liaise with clinicians, nurses and other health professionals
* Provide genetic advice for sample referrals and results
26
What is the difference between clinical validity and clinical utility?
* Clinical Validity: How well the test predicts the phenotype
* Clinical Utility: How the test adds to the management of the patient
27
What is the UKGTN?
* UK genetic testing network
* Body that approves all the genetic tests used in the UK
28
What are the 3 outcomes of a genetic test?
* **Pathogenic mutation**
* **Normal variation** (Polymorphism)
* **Novel variant** - Investigations needed to establish significance
29
How can you establish if a mutation is pathogenic?
* **Look at mode of inheritance and segregation of that mutation** by studying entire families
* **Look at Locus-specific databases of published and unpublished data**
* **Functional predictions of effect of mutation on gene function** - Nonsense, frameshift, splice site (exon+/-2 bp) mutations
* Missense/intronic mutation harder to prove if pathogenic as lots are tolerated within genome
* In-silico tools used for missense and splicing mutations
30
Use the following information from a case study of MFN2 to explain how the sibilings inherited this particular disease
Mitofusin 2 (MFN2) causes Charcot-Marie-Tooth disease type 2 (CMT2)
In one family there are two siblings with very severe early-onset CMT2 but parents unaffected
MFN2 sequenced by next generation sequencing: Both siblings homozygous for c.647T\>C p.(Phe216Ser)
Both parents should be heterozygous c.647T\>C p. but father is homozygous
* MLPA was used to measure dosage of all MFN2 exons in the family
* Found that children and father carry deletion of MFN2 exons 7-8
* This means children aren't homozygous for mutation instead they had one allele with the mutation and the other allele contained a deletion
31
How are the different variants found by Genomics England classified?
