29. Overview of Genomic Technologies in Clinical Diagnostics Flashcards
List some genetic technologies
- PCR
- Fragment analysis
- Sanger Sequencing
- Fluorescence in situ hybridisation (FISH)
- Array - comparative genomic hybridization (Array CGH)
- Multiplex ligation-dependent probe amplification (MLPA)
- Next-Generation sequencing
Describe polymerase chain reaction (PCR)
Fundamental for any DNA application
PCR is used to amplify a specific region of DNA; primers flank the region you want to amplify.
Each cycle doubles the amount of DNA copies of your target sequence
Amplify enough DNA molecules so that we have sufficient material to sequence or for other applications
How would you perform fragment analysis after PCR?
PCR based assay followed by capillary electrophoresis
Here we are sizing the PCR product
Can be used to detect repeat expansions or other small size changes (up to a few hundred bp)
Give an example of a repeat expansion disease that was diagnosed using fragment analysis
Huntington’s disease – severe neurodegenerative disorder
Caused by CAG repeat expansion in the Huntingtin (HTT) gene
Normal < 27 copies; Intermediate 27-35; Pathogenic > 35
Expanded protein is toxic and accumulates in neurons causing cell death
Diagnosed with fragment analysis
Briefly, describe sanger sequencing
Cycle Sequencing; based on the same principles as PCR
Each of the 4 DNA nucleotides has a different dye so we can determine the nucleotide sequence.
Here we are reading the dyes to obtain the DNA sequence. We can identify single nucleotide polymorphisms (SNPs), or mutations in this way
Can sequence up to 800 bp of sequence per reaction
Accurate (99.99%)
One reaction = one sequence
It is slow and low-throughput.
Costly to perform
What does FISH stand for, and what would you use it for?
Fluorescent in situ hybridisation
To detect large chromosomal abnormalities
Abnormalities include:
- Extra chromosomes
- Large deleted segments
- Translocations
How would you perform FISH?
1) Design Fluorescent probe to chromosomal region of interest
2) Denature probe and target DNA
3) Mix probe and target DNA (hybridisation)
4) Probe binds to target
5) Target fluoresces or lights up !
What is array CGH, and what is it used for?
An array comparative genomic hybridisation
Used for detection of sub-microscopic chromosomal abnormalities
Patient DNA labelled Green and control DNA labelled Red
Patient array comparative genomic hybridisation profile
Increased green signal over a chromosomal segment in the patient DNA
Indicates a gain in the patient sample not present in the parents
Describe how to perform an array CGH
STEPS 1-3: The patient and control DNA are labelled with fluorescent dyes and applied to the microarray.
STEP 4: The patient and control DNA compete to attach, or hybridise to the microarray
STEP 5: The microarray scanner measures the fluorescent signals
STEP 6: The computer software analyses the date and generates a plot.
The plot shows a patient array comparative hybridisation profile
An increased green signal over a chromosomal segment in the patient DNA indicates a gain in the patient sample that is not present in the parents
What is MLPA?
One probe oligonucleotide contains the sequence recognized by the forward primer, the other 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
What do we use MLPA for?
We use MLPA to detect abnormal copy numbers at specific chromosomal locations
MLPA can detect sub-microscopic (small) gene deletions/partial gene deletions
Perform fragment analysis of MLPA product
An important use of MLPA is to determine relative ploidy (how many chromosome copies?) as specific locations
For example, probes may be designed to target various regions of chromosome of a human cell
The signal strengths of the probes are compared with those obtained from a reference DNA sample known to have two copies of the chromosome
Describe the advancement in the technology of next generation sequencing
Technological advances since the end of the human genome project
Decrease in the cost of DNA sequencing
Development of new NGS methods began 13 years ago with 454 pyrosequencing
DNA sequencing throughput jumped 10 orders of magnitude
Solexa sequencing-by-synthesis (SBS) developed end of 2005
Sequencing market is now dominated by Illumina SBS sequencing
What is the current strategy with next generation sequencing?
An end to sequential testing
Wider range of tests in a shorter time for less money
Current strategy: Disease panels
Enriching to sequence only the known disease genes relevant to the phenotype
Panels expandable to include new genes as they are published
Potentially pathogenic variants confirmed by Sanger sequencing
Describe exome sequencing
There are ~21,000 genes in the human genome
Often we are only interested in the gene protein coding exons or ‘exome’ represents 1-2% of the genome
Some ~80% pathogenic mutations are protein coding
More efficient to only sequence the bits we are interested in, rather than the entire genome
Costs £1,000 for a genome, but only £200-£300 for an exome
Target enrichment
Capture target regions of interest with baits
Potential to capture several Mb genomic regions (typically 30-60 Mb)
Describe genome sequencing
Universally accepted that genome sequencing will become commonplace in diagnostic genetics
Not all tests will automatically move to whole exome sequencing / whole genome sequencing
- Panels/single gene tests may still be more suitable for some diseases.
- Capillary-based methods: Repeat expansions, MLPA, Family mutation Sanger sequencing
- Array-CGH: large sized chromosomal aberrations
What are some drawbacks of exome and genome sequencing?
Result interpretation is the greatest challenge
- 20,000 variants per coding genes ‘exome’
- Need for good variant databases of well phenotyped cases/control sequences
Ethical considerations
- Modified patient consent process
- Data analysis pathways – inspect relevant genes first
- Strategy for reporting ‘incidental’ findings
Infrastructure and training (particularly IT)
The NHS diagnositc lab do?
The main role of the lab is to help Consultants reach a genetic diagnosis for individuals and families to help guide treatment and management.
Perform specific tests with proven:
- Clinical Validity: How well the test predicts the phenotype
- Clinical Utility: How the test adds to the management of the patient
UKGTN (UK genetic testing network)-approved tests
In-depth and up-to-date knowledge of the genetic diseases covered
Translational Research
What are some requirements of an NHS diagnostic lab?
Accredited laboratory: ISO standard 15189 for Medical Laboratories.
Scientific, technical and administrative staff.
Provide clinical and laboratory diagnosis for inherited disorders.
Liaise with clinicians, nurses and other health professionals
Provide genetic advice for sample referrals and results
Carry out translational research for patient benefit
Train clinical research fellows, clinical scientists, post- and undergraduate students
List some of the different tests the NHS lab performs.
Diagnostic
- Diagnosis
- Management and Treatment
- Inform clinical trials
Family mutation
- Diagnosis
- Interpretation of pathogenicity
Predictive
- Life choices, management
Carrier (recessive)
- Life choices, management
Diagnostic testing is available for all Consultant referrals
- Clinical Geneticists most common referrers
Informed consent
- Genetic counselling
- Implications for other family members
What are some guidelines for diagnostic testing?
All referrals via Regional Genetics Centres
Close liaison with nurse specialists, genetic counsellors, clinicians during testing
International guidelines for predictive testing
Follow up at clinics, nurse led clinics, nurse telephone clinics as required
What are the possible outcomes from diagnostic test?
Pathogenic mutation
Normal variation
- Polymorphism
Novel variant
- Investigations to establish significance
Previously published/detected variant of uncertain significance
How would you interpret results from diagnostic testing?
- Which domain of protein affected?
- Segregation of mutation with disease in family in question
- De novo dominant
- If recessive, are they definitely on separate alleles – test parents
- Detected in other affected individuals? Age of onset?
- Has incomplete penetrance been described for this gene? - Amino-acid conservation between species
- Nucleotide conservation
- Transcript analysis
- Is the gene expressed in blood / fibroblasts?
- In-vitro splicing experiments - Functional studies
- E.g. Ion channel function in Xenopus oocytes
List some benefits of the 100,000 genome projects
- Bring direct benefit of genetics to patients
- Enable new scientific discovery and medical insights
- Create an ethical and transparent programme based on consent and patient engagement
- To kick-start the development of a UK genomics industry
- PERSONALISED MEDICINE
What was the 100,000 genome project?
- UK – wide collection
- GMCs (genomic medicine centres)
- Who/what is being sequenced?
- Rare diseases - families
- Cancer – germline and tumour samples
How were the results of the 100,000 genome project analysed using the genomics england panel app?
Genomics England Panel App
Community driven genetic interpretation
Crowdfunding research
‘Experts’ develop lists of possible genes than can cause a disease phenotype
These panels are reviewed by the community
Diseases have specific sets of virtual gene panels as a first port of call to look for pathogenic mutations
Thus we can focus on specific regions of the patients genome we think are important
What are the 3 classification of variant by genomics England?
Classification of variants by genomics England
Maximise diagnostic efficiency
Balance sensitivity and specificity
Variants within virtual panel divided into three tiers
- Tier 1 variants
- Known pathogenic
- Protein truncating - Tier 2 variants
- Protein altering (missense)
- Intronic (splice site) - Tier 3 variants
- Loss-of-function variants in genes not on the disease gene panel
