Overview of Genomic Technologies in Clinical Diagnostics Flashcards
List some examples of 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
What are the key points of Polymerase Chain Reaction (PCR)?
Fundamental for many DNA applications
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 for downstream applications
(Denaturation, Annealing, Extension)
What are the key points of Fragment Analysis?
PCR based assay
PCR followed by capillary electrophoresis (separating molecules by size)
Here we are sizing the PCR product
Can be used to detect repeat expansions or other small size changes (up to a few hundred base pairs)
What are some examples of Repeat Expansion Diseases?
Huntington’s disease – severe neurodegenerative disorder
Caused by CAG repeat expansion in the Huntingtin (HTT) gene
Normal < 27 copies; Intermediate 27-35 copies; Pathogenic > 35 copies
Expanded protein is toxic and accumulates in neurons causing cell death
Diagnosed with fragment analysis
What are the key points of 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.
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
reading the dyes to obtain the DNA sequence
We can identify single nucleotide polymorphisms (SNPs), or mutations
Detection of a mutation in a family by use of Sanger Sequencing
R1042G mutation in gene C3 segregates with affected individuals
Mutation causes disease cutaneous vasculitis
What are the key points of Fluorescence in situ hybridisation (FISH)?
To detect large chromosomal abnormalities
Extra chromosomes
Large deleted segments
Translocations
Chromosome abnormalities
What are the steps in FISH?
- 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 !
What are the key points to Array CGH?
Array comparative genomic hybridisation
For detection of sub-microscopic chromosomal abnormalities
Patient DNA labelled Green
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
What is Multiplex ligation-dependent probe amplification (MPLA) and what is it used for?
Multiplex ligation-dependent probe amplification (MLPA) is a variation of PCR that permits amplification of multiple targets
Each probe consists of two oligonucleotides which recognize adjacent target sites on the DNA
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 (capillary electrophoresis) 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
What does Next Generation Sequencing involve? (NGS)
What has NGS replaced?
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
Next Generation Sequencing has replaced Sanger sequencing for almost all sequencing tests in the lab
One of the most common NGS technique is EXOME SEQUENCING
What does Exome sequencing involve?
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
What is whole Genome sequencing?
NOT all tests will 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
What are some ethical considerations for exome and Genome Sequencing?
What is the greatest challenge?
Ethical considerations
Modified patient consent process
Data analysis pathways – inspect relevant genes first
Strategy for reporting ‘incidental’ findings
Result interpretation is the greatest challenge
20,000 genetic variants identified per coding genes ‘exome’
3 million variants in a whole human genome
Also when analysing findings etc, you need
Infrastructure and training (particularly IT and clinical scientists).
What is the The 100,000 Genomes Project?
100,000 genomes project
Bring direct benefit of whole genome sequencing and genetics to patients
Enable new scientific discovery and medical insights
Personalised medicine
England – wide collection GMCs (genomic medicine centres) Who/what is being sequenced? Rare diseases – index cases + families Cancer – germline and tumour samples
Classification of mutations by genomics England:
Variants within virtual panel divided into three tiers
(Expert review is required )
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
What is the NHS Diagnostic Laboratory and what does it do?
Accredited laboratory: ISO standard 15189 for Medical Laboratories
Scientific, technical and administrative staff
Provide clinical and laboratory diagnosis for genetic disorders
Liaise with clinicians, nurses and other health professionals
Provide genetic advice for sample referrals and results
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
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
