Clinical Genetics: Overview of genomic technologies in clinical diagnostics Flashcards
Give a brief overview of what PCR (polymerase chain reaction) is and how it works
- 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
What is fragment analysis?
- 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
What can fragment analysis be used for?
- 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
What is Huntingdon’s disease?
- It is a severe neurodegenerative disorder caused by CAG repeat expansion in the Huntingtin (HTT) gene
How does the CAG expansion in the Huntingtin gene causes Huntingdon’s disease?
- 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
How is Huntingdon’s disease diagnosed?
- Diagnosed with fragment analysis
Give a brief overview of sanger sequencing
- 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
What are some of the disadvantages of sanger sequencing?
- Slow
- Low-throughput
- Costly to perform for large numbers of samples
What can sanger sequencing be used for?
- Can be used to identify single nucleotide polymorphisms (SNPs), or mutations
Give a brief overview of Fluoresence in situ hybridisation (FISH)
- Used to microscopically detect large chromosomal abnormalities such as:
- Extra chromosomes
- Large deleted segments
- Translocations
How does fluoresence in situ hybridisation work?
- 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
Give a brief overview of Array CGH (comparative genomic hybridisation)
- Used for detection of sub-microscopic chromosomal abnormalities
- Patient DNA labelled Green
- Control DNA labelled Red
How does Array CGH work?
- 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
What does each colour of fluoresence on the microarray for array CGH represent?
- 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
What is MLPA?
- 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
How does MLPA work?
- 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
Why is MLPA an example of a quantitative assay?
- Because the amount of MLPA product produced is proportional to the amount of starting DNA
How is the data from MLPA analysed?
- 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
Why has next generation sequencing replaced sanger sequencing for almost all sequencing tests?
- Higher DNA sequencing throughput
- Provides a wider range of tests in a shorter time for less money
Why is whole exome sequencing used more often tha whole genome sequencing?
- 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
How does whole exome sequencing work?
- 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
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?
- 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
What is the main disadvantage of Whole genome sequencing currently?
- 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
What are some other disadvantages of whole genome sequencing?
- 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
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
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
What is the UKGTN?
- UK genetic testing network
- Body that approves all the genetic tests used in the UK
What are the 3 outcomes of a genetic test?
- Pathogenic mutation
- Normal variation (Polymorphism)
- Novel variant - Investigations needed to establish significance
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
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
How are the different variants found by Genomics England classified?
