Overview of genomic technologies in clinical diagnostics Flashcards
List 7 Genomic 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
Polymerase Chain Reaction (PCR) - use
PCR is used to amplify a specific region of DNA
Polymerase Chain Reaction (PCR) - action
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 for downstream applications
Fragment analysis - define
PCR based assay
PCR followed by capillary electrophoresis
Here we are sizing the PCR product
Fragment analysis - use
Can be used to detect repeat expansions or other small size changes (up to a few hundred bp)
Huntington’s disease - define
Huntington’s disease – severe neurodegenerative disorder
Huntington’s disease - cause
Caused by CAG repeat expansion in the Huntingtin (HTT) gene
Expanded protein is toxic and accumulates in neurons causing cell death
Huntington’s disease - ranges of normal to pathogenic
Normal < 27 copies; Intermediate 27-35 copies; Pathogenic > 35 copies
Huntington’s disease - diagnosed using
Diagnosed with fragment analysis
Sanger Sequencing - define
Cycle Sequencing; based on the same principles as PCR
Sanger Sequencing - describe action
Each of the 4 DNA nucleotides has a different dye so we can determine the nucleotide sequence.
Sanger Sequencing - +ves/-ves
Up to 800bp of sequence per reaction
- Good for sequencing single exons of genes
Slow, low-throughput and costly to perform for large numbers of samples
FISH - to detect
To detect large chromosomal abnormalities
Extra chromosomes
Large deleted segments
Translocations
FISH - define
Fluorescent in situ hybridisation
Uses fluorescent probes binding parts of the chromosome to show a high degree of sequence complementarity
FISH - describe method
Design Fluorescent probe to chromosomal region of interest
Denature probe and target DNA
Mix probe and target DNA (hybridisation)
Probe binds to target
Target fluoresces or lights up !
Array CGH - define
Array comparative genomic hybridisation
ultra-high resolution way of objectively and quantitatively detecting. whether a patient’s DNA has losses (deletions) or gains (duplications, triplications. etc) which are pathogenic
Array CGH - use
For detection of sub-microscopic chromosomal abnormalities
Array CGH - results
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
MLPA - define
Multiplex ligation-dependent probe amplification (MLPA) is a variation of PCR that permits amplification of multiple targets
MLPA - action
Each probe consists of two oligonucleotides which recognize adjacent target sites on the DNA
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
MLPA - use
We use MLPA to detect abnormal copy numbers at specific chromosomal locations
MLPA can detect sub-microscopic (small) gene deletions/partial gene deletions
MLPA - describe action of the product
Perform fragment analysis (capillary electrophoresis) of MLPA product
MLPA - describe involvement in ploidy
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
Explain the 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
Explain reason for using 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
Exome Sequencing - briefly describe action
Target enrichment
Capture target regions of interest with baits - streptavidin coated
Exome Sequencing - capacity for results
Potential to capture several Mb genomic regions (typically 30-60 Mb
Describe which tests will not automatically move to whole genome sequencing
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
Explain the challenge of result interpretation in sequencing
Result interpretation is the greatest challenge
20,000 variants per coding genes ‘exome’
3 million variants in a whole human genome
Explain the ethical considerations in sequencing
Ethical considerations:
Modified patient consent process
Data analysis pathways – inspect relevant genes first
Strategy for reporting ‘incidental’ findings
Describe a challenge of genome/exome sequencing
Infrastructure and training (particularly IT and clinical scientists)
The NHS Diagnostic Laboratory - main role
The main role of the lab is to help Consultants reach a genetic diagnosis for individuals and families to help guide treatment and clinical management
The NHS Diagnostic Laboratory - perform specific tests with proven what
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
The NHS Diagnostic Laboratory - list 5 roles
Diagnostic (diagnosis, treatment, pathogenicity)
Predictive (life choices)
Carrier (recessive)
Informed consent (counselling/implications)
Diagnostic testing is available for all Consultant referrals
Diagnostic Testing - list regulations
All referrals via Regional Genetics Centres
Close liaison with nurse specialists, genetic counsellors, clinicians during testing
Strict international guidelines for predictive testing
Follow up at clinics, nurse led clinics, nurse telephone clinics as required
Diagnostic Test Outcomes - list
Pathogenic mutation
Normal variation
Polymorphism
Novel variant
Investigations to establish significance
How to establish if a mutation is pathogenic?
Mode of inheritance
Locus-specific databases of published and unpublished data
Nonsense, frameshift, splice site (exon+/-2 bp) mutations
Missense/intronic mutation
- In-silico tools for missense and splicing mutations
Interpreting Results - list regu;ations
Do not report known polymorphisms
Conservative approach to reporting novel mutations of uncertain pathogenicity
- ‘Uncertain significance’
- ‘Likely to be pathogenic’
Request samples from family members
Continue testing other genes
100,000 genomes project - define
UK Government project that is sequencing whole genomes from National Health Service patients
100,000 genomes project - +ves
100,000 genomes project:
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
Personalised medicine
100,000 genomes project - Who/what is being sequenced?
Rare diseases – index cases + families
Cancer – germline and tumour samples
Explain how the use of the Genomics England Panel App allows us to focus on specific genes in pt’s genome we think are important
Genomics England Panel App
‘Experts’ develop lists of possible genes than can cause a specific disease
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 genes of the patients genome we think are important
Compare Tier 1-3 variants
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
Describe the formation of the Tier 1-3 variant classification system
Classification of mutations by genomics England
Variants within virtual panel divided into three tiers
Expert review is required
