Overview Of Genomic Technologies for Clinical Diagnosis Flashcards
- Briefly explain what PCR does?
• Used to amplify a specific region of DNA for downstream applications – each cycle doubles the DNA amount.
• Primers flank the region you want to amplify
Process : Denaturation , Annealing , Extension
- Explain how PCR works?
- PCR based assay - PCR followed by capillary electrophoresis to size the PCR product
- Can be used to detect repeat expansions or other small size changes (up to a few hundred bp).
- What is the repeat expansion disease Huntingtons?
•Huntington’s disease (severe neurodegenerative disorder) is caused by CAG repeat expansion in the Huntingtin (HTT) gene.
- How many copies of the CAG codon in a normal, intermediate and pathogenic situation?
Normal < 27 copies
Intermediate 27-35 copies
Pathogenic > 35 copies
•Accumulation of expanded toxic protein in neurons causes cell death.
- How does Sanger Sequencing work?
•Each of the 4 DNA nucleotides are labelled with a different dye - determine the nucleotide sequence to look for mutations or single nucleotide polymorphisms (SNPs).
- Is sanger sequencing good for sequencing large numbers or samples or only single exons?
•Up to 800bp of sequence per reaction so it’s good for sequencing single exons of genes but slow, low-throughput and costly to perform for large numbers of samples
- Whats an example of a mutation that causes the disease cutaneous vasculitis ?
R1042G mutation in gene C3 segregates with affected individuals and causes disease cutaneous vasculitis
- What is FISH used to detect?
- Detect large chromosomal abnormalities e.g. extra chromosomes, large deleted segments and translocations.
- Cultured cells, metaphase spread - Microscopic (5-10Mb)
- How does FISH work?
- Design a fluorescent probe to the chromosomal region of interest, denature target DNA and mix probe with it until hybridisation occurs (probe binds to target causing it to fluoresce/light up).
- Can use it for the whole karyotype or a specific chromosome.
- What is Array CGH (comparative genomic hybridisation) used to detect?
• For detection of sub-microscopic chromosomal abnormalities as well as large chromosomal abnormalities.
- How does Array CGH work?
* GO through this properly*
- The patient DNA is labelled green and control DNA is labelled red. They are mixed and allowed to hybridise to an array before scanning to produce a patient array comparative genomic hybridisation profile.
- Increased green signal over a chromosomal segment in the patient DNA indicates a gain in the patient sample chromosomes that is not present in the parents.
- What is a MLPA (Multiplex ligation-dependent probe amplification) used to detect?
• Variation of PCR that permits amplification of multiple targets – used to detect abnormal copy numbers at specific chromosomal locations (relative ploidy) and sub-microscopic (small) gene deletions/partial gene deletions.
- How does an MLPA work?
• Probes consist of two oligonucleotides that recognize adjacent target sites on the DNA – one oligonucleotide is recognised by the forward primer and the other is recognised by the reverse primer.
o When both oligonucleotides hybridize to their respective targets - they can be ligated into a complete probe.
• Perform fragment analysis (capillary electrophoresis) of MLPA product.
• 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 has Sanger Sequencing been replaced by?
Next Generation Sequencing
- Which techniques are used in next gen sequencing?
- Solexa sequencing-by-synthesis (SBS) developed end of 2005
* Sequencing market is now dominated by Illumina SBS sequencing
- Whats the major advantage of next generation sequencing?
Wider range of tests in a shorter time for less money (through-put jumped 10 orders of magnitude).
- One current strategy for Next Gen sequencing is “Disease Panel Tests “ how do these works?
o Sequences are enriched for known disease genes relevant to the phenotype and expanded to include new genes as they are published.
o Potentially pathogenic variants confirmed by Sanger sequencing.
- What is useful about exome sequencing?
- Often we are only interested in the exome because most pathogenic mutations (80%) are usually protein coding and it is 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
- How does exome sequencing work?
• Target enrichment allows us to capture specific target regions:
o RNA baits and magnetic beads tagged with biotin (or other markers) that bind to the baits are used to capture specific regions of interest.
o Potential to capture several Mb genomic regions (typically 30-60 Mb)
- What is the future for whole genome sequencing?
• WGS will become commonplace in diagnostic genetics in the future but not all tests will automatically move to whole genome sequencing because some diseases are more suitable to other methods:
- Which diseases may not be suitable for whole genome sequencing -> What would it be preferable to use ?
o Panels/single gene tests may still be more suitable for some diseases, e.g. cystic fibrosis
o Capillary-based methods: Repeat expansions, MLPA, family mutation confirmation Sanger sequencing
o Array-CGH: large sized chromosomal aberrations
- Is whole genome sequencing easy/quick?
• Data generation and processing are relatively automated/quick but data interpretation of clinical genomes currently has a substantial manual component (most tricky part).
- How many variants are there is a coded exome and in a whole human genome?
20,000 variants per coding genes ‘exome’ and 3 million variants in a whole human genome.
- What ethical considerations need to be considered when exome and genome sequencing?
o Modified patient consent process
o Data analysis pathways – inspect relevant genes first
o Strategy for reporting ‘incidental’ findings
- What is the NHS diagnostics laboratory?
• Accredited laboratory with scientific, technical and administrative staff that provides clinical/laboratory diagnosis for genetic disorders and provides genetic advice for sample referrals/results.
- What is the main role of the NHS diagnostics laboratory?
help consultants reach a genetic diagnosis s to help guide treatment and clinical management
•Perform specific tests with proven :
-clinical validity (how well the test predicts the phenotype) and
-clinical utility (how the test adds to the management of the patient).
- Where could you find up to date in depth knowledge of genetic diseases
•UKGTN (UK genetic testing network)-approved
- What 4 main uses are there of the NHS diagnostic laboratory?
o Diagnostic: Diagnosis, Management and Treatment and Interpretation of pathogenicity
o Predictive: Life choices, management
o Carrier (recessive): Life choices, management
o Informed consent: Genetic counselling and Implications for other family members
- Are all diagnostic testing referrals via Regional Genetics Centres?
Yes
- Who does the patient have close contact with during diagnostic testing?
• Close liaison with nurse specialists, genetic counsellors, clinicians during testing - follow up at clinics, nurse led clinics, nurse telephone clinics as required.
- What can be found during diagnostic testing?
pathogenic mutations, normal variants (polymorphism) and novel variants (investigation required for significance).
- How do we establish if a mutation is pathogenic?
- Mode of inheritance - Request samples from family members.
- Locus-specific databases of published and unpublished data
- Missense/intronic mutation: In-silico tools for missense and splicing mutations
- Conservative approach to reporting novel mutations of uncertain pathogenicity - ‘Uncertain significance’ or ‘Likely to be pathogenic’
- What is Charcot-Marie-Tooth disease type 2 (CMT2)
o Degeneration of the long nerves in legs and arms leading to muscle wasting and sensory defects.
o Onset usually in childhood
- Which mutation causes Charcot-Marie-Tooth disease type 2 (CMT2)
•Mitofusin 2 (MFN2)
- Case study:
• Two siblings with very severe early-onset CMT2 and parents unaffected.
• NGS: Gene sequenced and found to be homozygous for c.647T>C p.(Phe216Ser).
• Parents sequenced, expected them to both be heterozygous but the father was homozygous TT.
Explain why this could be?
o Could be due to non-mendelian inheritance, father not the father, paternal exon deletion etc.
- Which exon deletions were found in both father and son after MLPA?
- MLPA measures dosage of all MFN2 exons and found that the child and father carry MFN2 exons 7-8 deletions.
- Breakpoint sequencing: Deletion of 1,476 bp from 700 bp 3’ of ex 6 to 2.1kb 5’ of ex 9
- What is the use of genomic england (a 100,000 genome 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 and personalised medicine.
- How do healthcare professionals interpret genomic testing clinically?
- ‘Experts’ develop lists of possible genes than can cause a specific disease - are reviewed by the community.
- Diseases have specific sets of virtual gene panels as a first port-of-call to look for pathogenic mutations so we can focus on specific genes of the patient’s genome we think are important
- • Classification of mutations by genomics England - variants within virtual panel divided into three tiers- Explain these?
o Tier 1 variants: Known pathogenic or Protein truncating.
o Tier 2 variants: Protein altering (missense) or Intronic (splice site).
o Tier 3 variants: Loss-of-function variants in genes not on the disease gene panel.
