Overview of Genomic Techniques in Diagnostics Flashcards
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)
- 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
Fragment Analysis
- PCR based assay
- PCR 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)
Repeat Expansion Diseases I
- 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
Sanger Sequencing pt 1.
- 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
Sanger Sequencing pt 2.
- Remember your FTO Gene sequencing practical ??
- Here we are 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
FISH - Fluorescent in situ hybridisation intro
- 1969, Gall & Pardue
- Cultured cells, metaphase spread
- Microscopic (5-10Mb)
- To detect large chromosomal abnormalities
- Extra chromosomes
- Large deleted segments
- Translocations
FISH pt2.
- 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
- 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
MLPA pt1.
• 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
• 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 pt2.
• 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
Next Generation Sequencing
• An end to sequential testing -Next Generation Sequencing has replaced Sanger sequencing for almost all sequencing tests in the lab
• 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
Exome Sequencing
- Target enrichment
- Capture target regions of interest with baits
- Potential to capture several Mb genomic regions (typically 30-60 Mb
- 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
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
Interpretation of clinical genomes currently has a substantial manual component
Whole genome sequencing is NOT trivial
diagrams
Exome and Genome Sequencing
• Result interpretation is the greatest challenge
• 20,000 genetic variants identified per coding genes ‘exome’
• 3 million variants in a whole human genome
• Ethical considerations
- Modified patient consent process
- Data analysis pathways – inspect relevant genes first
- Strategy for reporting ‘incidental’ findings
• Infrastructure and training (particularly IT and clinical
scientists)
Genomics England I
• 100,000 genomes project • Bring direct benefit of whole genome sequencing and genetics to patients • Enable new scientific discovery and medical insights • Personalised medicine
Genomics England II
- England – wide collection
- GMCs (genomic medicine centres)
- Who/what is being sequenced?
- Rare diseases – index cases + families
- Cancer – germline and tumour samples
Clinical Interpretation II
- 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 pane
The NHS Diagnostic Laboratory pt1.
- 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 NHS Diagnostic Laboratory pt2.
• 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
The NHS Diagnostic Laboratory pt3.
• Diagnostic
- Diagnosis
- Management and Treatment
- 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
Diagnostic Test Outcomes
• Pathogenic mutation
• Normal variation
- Polymorphism
• Novel variant
- Investigations to establish clinical significance…
How to establish if a mutation is pathogenic?
- Mode of inheritance
- Genetic 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
• 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 ?
Case Example: MFN2 pt1.
• Mitofusin 2 (MFN2) causes Charcot-Marie-Tooth disease type 2 (CMT2)
– Degeneration of the long nerves in legs and arms leading to muscle wasting and sensory defects.
– Onset usually in childhood
– Autosomal Dominant and Autosomal Recessive
• Two siblings with very severe early-onset CMT2.
• Parents unaffected
• MFN2 sequenced by next generation sequencing
– Apparently homozygous for c.647T>C p.(Phe216Ser) mutation
– Parents sequenced, expected them to both be heterozygous
Case Example: MFN2 pt2.
c.647T>C p.(Phe216Ser)
Mutation:
sanger sequencing/ validation
father appeared homozygous WT, not heterozygous as was expected
Deletion of MFN2 Exons 7-8
MLPA of MFN2 gene exons:
• MLPA measures dosage of all MFN2 exons
• Affected children carry deletion of MFN2 exons 7-8 inherited from father
Case Example: MFN2
diagram
